RNA containing modified nucleosides and methods of use thereof

ABSTRACT

This invention provides RNA, oligoribonucleotide, and polyribonucleotide molecules comprising pseudouridine or a modified nucleoside, gene therapy vectors comprising same, methods of synthesizing same, and methods for gene replacement, gene therapy, gene transcription silencing, and the delivery of therapeutic proteins to tissue in vivo, comprising the molecules. The present invention also provides methods of reducing the immunogenicity of RNA, oligoribonucleotide, and polyribonucleotide molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of and claims priority to U.S.patent application Ser. No. 11/990,646, filed Mar. 27, 2009, which is a35 U.S.C. §371 national phase application from, and claiming priorityto, International Application No. PCT/US06/32372, filed Aug. 21, 2006,all of which applications are hereby incorporated by reference in theirentireties herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numbersAI060505, AI50484, and DE14825 awarded by the National Institutes ofHealth. The Government therefore has certain rights in this invention.

FIELD OF INVENTION

This invention provides RNA, oligoribonucleotide, and polyribonucleotidemolecules comprising pseudouridine or a modified nucleoside, genetherapy vectors comprising same, methods of synthesizing same, andmethods for gene replacement, gene therapy, gene transcriptionsilencing, and the delivery of therapeutic proteins to tissue in vivo,comprising the molecules. The present invention also provides methods ofreducing the immunogenicity of RNA, oligoribonucleotide, andpolyribonucleotide molecules.

BACKGROUND OF THE INVENTION

All naturally occurring RNA is synthesized from four basicribonucleotides ATP, CTP, UTP and GTP, but some of the incorporatednucleosides are modified post-transcriptionally in almost all types ofRNA. Nearly one hundred different nucleoside modifications have beenidentified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). TheRNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197). Theextent and nature of modifications vary and depend on the RNA type aswell as the evolutionary level of the organism from where the RNA isderived. Ribosomal RNA, the major constituent of cellular RNA, containssignificantly more nucleoside modifications in mammalian cells thanbacteria. Human rRNA, for example, has 10-times more pseudouridine (Ψ)and 25-times more 2′-O-methylated nucleosides than bacterial rRNA, whilerRNA from mitochondria has very few modifications. Transfer RNA (tRNA)is the most heavily modified subgroup of RNA. In mammalian tRNA, up to25% of the nucleosides are modified, while prokaryotic tRNA containssignificantly fewer modifications. Bacterial messenger RNA (mRNA)contains no nucleoside modifications, while mammalian mRNA containsmodified nucleosides such as 5-methylcytidine (m⁵C), N6-methyladenosine(m⁶A), inosine and 2′-O-methylated nucleosides, in addition toN7-methylguanosine (m⁷G), which is part of the 5′-terminal cap. The roleof nucleoside modifications on the immuno-stimulatory potential and onthe translation efficiency of RNA, however, is not known.

SUMMARY OF THE INVENTION

This invention provides RNA, oligoribonucleotide, and polyribonucleotidemolecules comprising pseudouridine or a modified nucleoside, genetherapy vectors comprising same, gene therapy methods and genetranscription silencing methods comprising same, methods of reducing animmunogenicity of same, and methods of synthesizing same.

In one embodiment, the present invention provides a messenger RNAcomprising a pseudouridine residue.

In another embodiment, the present invention provides an RNA moleculeencoding a protein of interest, said RNA molecule comprising apseudouridine residue.

In another embodiment, the present invention provides an invitro-transcribed RNA molecule, comprising a pseudouridine or a modifiednucleoside.

In another embodiment, the present invention provides an invitro-synthesized oligoribonucleotide, comprising a pseudouridine or amodified nucleoside, wherein the modified nucleoside is m⁵C, m⁵U, m⁶A,s²U, Ψ, or 2′-O-methyl-U.

In another embodiment, the present invention provides a gene-therapyvector, comprising an in vitro-synthesized polyribonucleotide molecule,wherein the polyribonucleotide molecule comprises a pseudouridine or amodified nucleoside.

In another embodiment, the present invention provides a double-strandedRNA (dsRNA) molecule containing, as part of its sequence, apseudouridine or a modified nucleoside and further comprising an siRNAor shRNA. In another embodiment, the dsRNA molecule is greater than 50nucleotides in length. Each possibility represents a separate embodimentof the present invention.

In another embodiment, the present invention provides a method forinducing a mammalian cell to produce a recombinant protein, comprisingcontacting the mammalian cell with an in vitro-synthesized RNA moleculeencoding the recombinant protein, the in vitro-synthesized RNA moleculecomprising a pseudouridine or a modified nucleoside, thereby inducing amammalian cell to produce a recombinant protein.

In another embodiment, the present invention provides a method fortreating anemia in a subject, comprising contacting a cell of thesubject with an in vitro-synthesized RNA molecule, the invitro-synthesized RNA molecule encoding erythropoietin, thereby treatinganemia in a subject.

In another embodiment, the present invention provides a method fortreating a vasospasm in a subject, comprising contacting a cell of thesubject with an in vitro-synthesized RNA molecule, the invitro-synthesized RNA molecule encoding inducible nitric oxide synthase(iNOS), thereby treating a vasospasm in a subject.

In another embodiment, the present invention provides a method forimproving a survival rate of a cell in a subject, comprising contactingthe cell with an in vitro-synthesized RNA molecule, the invitro-synthesized RNA molecule encoding a heat shock protein, therebyimproving a survival rate of a cell in a subject.

In another embodiment, the present invention provides a method fordecreasing an incidence of a restenosis of a blood vessel following aprocedure that enlarges the blood vessel, comprising contacting a cellof the blood vessel with an in vitro-synthesized RNA molecule, the invitro-synthesized RNA molecule encoding a heat shock protein, therebydecreasing an incidence of a restenosis in a subject.

In another embodiment, the present invention provides a method forincreasing a hair growth from a hair follicle is a scalp of a subject,comprising contacting a cell of the scalp with an in vitro-synthesizedRNA molecule, the in vitro-synthesized RNA molecule encoding atelomerase or an immunosuppressive protein, thereby increasing a hairgrowth from a hair follicle.

In another embodiment, the present invention provides a method ofinducing expression of an enzyme with antioxidant activity in a cell,comprising contacting the cell with an in vitro-synthesized RNAmolecule, the in vitro-synthesized RNA molecule encoding the enzyme,thereby inducing expression of an enzyme with antioxidant activity in acell.

In another embodiment, the present invention provides a method fortreating cystic fibrosis in a subject, comprising contacting a cell ofthe subject with an in vitro-synthesized RNA molecule, the invitro-synthesized RNA molecule encoding Cystic Fibrosis TransmembraneConductance Regulator (CFTR), thereby treating cystic fibrosis in asubject.

In another embodiment, the present invention provides a method fortreating an X-linked agammaglobulinemia in a subject, comprisingcontacting a cell of the subject with an in vitro-synthesized RNAmolecule, the in vitro-synthesized RNA molecule encoding a Bruton'styrosine kinase, thereby treating an X-linked agammaglobulinemia.

In another embodiment, the present invention provides a method fortreating an adenosine deaminase severe combined immunodeficiency (ADASCID) in a subject, comprising contacting a cell of the subject with anin vitro-synthesized RNA molecule, the in vitro-synthesized RNA moleculeencoding an ADA, thereby treating an ADA SCID.

In another embodiment, the present invention provides a method forproducing a recombinant protein, comprising contacting an in vitrotranslation apparatus with an in vitro-synthesized polyribonucleotide,the in vitro-synthesized polyribonucleotide comprising a pseudouridineor a modified nucleoside, thereby producing a recombinant protein.

In another embodiment, the present invention provides a method ofsynthesizing an in vitro-transcribed RNA molecule comprising a modifiednucleotide with a pseudouridine modified nucleoside, comprisingcontacting an isolated polymerase with a mixture of unmodifiednucleotides and the modified nucleotide.

In another embodiment, the present invention provides an in vitrotranscription apparatus, comprising: an unmodified nucleotide, anucleotide containing a pseudouridine or a modified nucleoside, and apolymerase. In another embodiment, the present invention provides an invitro transcription kit, comprising: an unmodified nucleotide, anucleotide containing a pseudouridine or a modified nucleoside, and apolymerase. Each possibility represents a separate embodiment of thepresent invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Production of TNF-α by MDDCs transfected with natural RNA,demonstrating that unmodified in vitro-synthesized RNA and bacterial andmitochondrial RNA is highly immunogenic, while other mammalian RNA isweakly immunogenic. Human MDDCs were incubated with Lipofectin® alone,or complexed with R-848 (1 μg/ml), or RNA (5 μg/ml) from 293 cells(total, nuclear and cytoplasmic RNAs), mouse heart (polyA+ mRNA), humanplatelet mitochondrial RNA, bovine tRNA, bacterial tRNA and total RNA(E. coli) with or without RNase digestion. After 8 h, TNF-α was measuredin the supernatants by ELISA. Mean values±SEM are shown. Results arerepresentative of 3 independent experiments.

FIG. 2. TLR-dependent activation by RNA demonstrates that m6A and s2Umodification blocks TLR3 signaling, while all modifications block TLR7and TLR8 signaling, and that less modified bacterial RNA and unmodifiedin vitro-transcribed RNA activates all three TLR. (A) Aliquots (1 μg) ofin vitro-transcribed RNA-1571 without (none) or with m⁵C, m⁶A, Ψ, m⁵U ors²U nucleoside modifications were analyzed on denaturing agarose gelfollowed by ethidium bromide-staining and UV-illumination. (B) 293 cellsexpressing human TLR3, TLR7, TLR8 and control vectors were treated withLipofectin® alone, Lipofectin®-R-848 (1 μg/ml) or RNA (5 μg/ml).Modified nucleosides present in RNA-730 and RNA-1571 are noted.293-ELAM-luc cells were use as control cells. (C) CpG ODN-2006 (5μg/ml), LPS (1.0 μg/ml) and RNA isolates were obtained from rat liver,mouse cell line (TUBO) and human spleen (total), human plateletmitochondrial RNA, or from two different E. coli sources. 293-hTLR9cells served as control. After 8 h, IL-8 was measured in thesupernatants by ELISA. Mean values±SEM are shown. Cell lines containinghTLR3-targeted siRNA are indicated with asterisk. The results arerepresentative of four independent experiments.

FIG. 3. Cytokine production by RNA-transfected DC demonstrates that allmodifications block activation of cytokine generated DC, while onlyuridine modifications block blood-derived DC activation. MDDC generatedwith GM-CSF/IL-4 (A, C) or GM-CSF/IFN-α MDDCs (B), and primary DC1 andDC2 (D) were treated for 8 to 16 h with Lipofectin® alone,Lipofectin®-R-848 (1 μg/ml) or RNA (5 μg/ml). Modified nucleosidespresent in RNA-1571 are noted. TNF-α, IL-12(p70) and IFN-α were measuredin the supernatant by ELISA. Mean values±SEM are shown. The results arerepresentative of 10 (A and C), 4 (B), and 6 (D) independentexperiments. E. Activation of DC by RNA. MDDC were treated for 20 h withLipofectin® alone or complexed with 1 μg/ml poly(I):(C) or R-848 aspositive controls (top panel) or Lipofectin® complexed with theindicated RNA (5 μg/ml; bottom panel). Modified nucleosides present inRNA-1886 are noted. TNF-α was measured in the supernatants by ELISA.Expression of CD83, CD80, and HLA-DR was determined by flow cytometry.

FIG. 4. Activation of DC by RNA demonstrates that all modificationsinhibit DC activation. MDDC were treated for 20 h with Lipofectin®alone, Lipofectin®-R-848 (1 μg/ml) or RNA-1571, modified as indicated (5μg/ml). (A) CD83 and HLA-DR staining (B) TNF-α levels in thesupernatants and mean fluorescence of CD80 and CD86 in response toincubation with RNA. The volume of medium was increased 30-fold for flowcytometry, as indicated by the asterisk. Data are representative of fourindependent experiments.

FIG. 5. Capped RNA-1571 containing different amounts (0, 1, 10, 50, 90,99 and 100% of modified nucleoside, relative to the correspondingunmodified NTP) were transcribed, and it was found that modification ofonly a few nucleosides resulted in an inhibition of activation of DC. A.All transcripts were digested to monophosphates and analyzed byreversed-phase HPLC to determine the relative amount of modifiednucleoside incorporation. Representative absorbance profiles obtained atthe indicated (Ψ:U) ratios are shown. Elution times are noted for3′-monophosphates of pseudouridine (Ψ), cytidine (C), guanosine (G),uridine (U), 7-methylguanosine (“m7G”) and adenosine (“A”). (B) Modifiednucleoside content of RNA-1571. The expected percentage of m⁶A, Ψ or m⁵Cin RNA-1571 was calculated based on the relative amount of modified NTPin the transcription reaction and the nucleoside composition of RNA-1571(A: 505, U: 451, C: 273, G: 342). Values for measured modifiednucleoside content were determined based on quantitation of the HPLCchromatograms. Notes: A: values (%) for m6ATP, ΨTP and m⁵CTP relative toATP UTP and CTP, respectively. B: values for m⁶A, Ψ and m⁵Cmonophosphates relative to all NMPs. (C) MDDC were transfected withLipofectin®-complexed capped RNA-1571 (5 μg/ml) containing the indicatedamount of m6A, Ψ or m⁵C. After 8 h, TNF-α was measured in thesupernatants. Data expressed as relative inhibition of TNF-α. Meanvalues±SEM obtained in 3 independent experiments are shown.

FIG. 6. TNF-α expression by oligoribonucleotide-transfected DCsdemonstrates that as few as one modified nucleoside reduces DCactivation. (A) Sequences of oligoribonucleotides (ORN) synthesizedchemically (ORN1-4) or transcribed in vitro (ORN5-6) are shown.Positions of modified nucleosides Um (2′-O-methyluridine), m⁵C and Ψ arehighlighted. Human MDDC were transfected with Lipofectin® alone(medium), R-848 (1 μg/ml) or Lipofectin® complexed with RNA (5 μg/ml).Where noted, cells were treated with 2.5 μg/ml cycloheximide (CHX). (B).After 8 h incubation, TNF-α was measured in the supernatant. (C) RNAfrom the cells was analyzed by Northern blot. Representative meanvalues±SEM of 3 independent experiments are shown.

FIG. 7. A. ΨmRNA does not stimulate pro-inflammatory cytokine productionin vivo. Serum samples (6 h after injection) were analyzed by ELISA andrevealed that 3 μg of unmodified mRNA induced a higher level of IFN-αthan did 3 μg of ψ-modified mRNA (P<0.001). Levels of IFN-α induced by 3μg of ψ-modified mRNA were similar to those obtained when animals wereinjected with uncomplexed lipofectin. Values are expressed as themean±s.e.m. (n=3 or 5 animals/group). B. Similar results were observedwith TNF-α.

FIG. 8. mRNA containing pseudouridine (Ψ) does not activate PKR. Ψ:pseudouridine. Control: unmodified RNA. m5C: mRNA with m⁵C modification.

FIG. 9. Increased expression of luciferase from pseudouridine-containingmRNA in rabbit reticulocyte lysate. Luc-Y: mRNA with pseudouridinemodification; luc-C: unmodified RNA. Data is expressed by normalizingluciferase activity to unmodified luciferase RNA.

FIG. 10. Increased expression of renilla from pseudouridine-containingmRNA in cultured cells. A. 293 cells. B. Murine primary, bonemarrow-derived mouse dendritic cells. renilla-Y: mRNA with pseudouridinemodification; renilla-C: unmodified RNA. RNA was modified with m⁵C, m⁶A,and m⁵U as noted.

FIG. 11. A. Additive effect of 3′ and 5′ elements on translationefficiency of ψmRNA. 293 cells were transfected with firefly luciferaseconventional and ψmRNAs that had 5′ cap (capLuc), 50 nt-long 3′polyA-tail (TEVlucA50), both or neither of these elements (capTEVlucA50and Luc, respectively). Cells were lysed 4 h later and luciferaseactivities measured in aliquots ( 1/20th) of the total lysates. B. ψmRNAis more stable than unmodified mRNA. 293 cells transfected withcapTEVlucA_(n) containing unmodified or ψ-modified nucleosides werelysed at the indicated times following transfection. Aliquots ( 1/20th)of the lysates were assayed for luciferase. Standard errors are toosmall to be visualized with error bars. C. Expression of β-galactosidaseis enhanced using ψmRNA compared with conventional mRNA. 293 cellsseeded in 96-well plates were transfected with lipofectin-complexedmRNAs (0.25 μg/well) encoding bacterial β-galactosidase (lacZ). Thetranscripts had cap and 3′ polyA-tail that were either 30 nt-long(caplacZ) or ˜200 nt-long (caplacZ-An). Constructs made usingconventional U or ψ nucleosides were tested. Cells were fixed andstained with X-gal, 24 h post-transfection. Images were taken byinverted microscopy (40 and 100× magnification) from representativewells.

FIG. 12. A. Expression of renilla following intracerebral injection ofmodified or unmodified encoding mRNA. Rat brain cortex was injected at 8sites/animals. One hemisphere was injected with capped, renilla-encodingRNA with pseudouridine modification (capRenilla-Y), while thecorresponding hemisphere with capped RNA with no nucleoside modification(capRenilla-C). Data from 2 animals (6 injection sites) are shown. BG;lower level of detection of the assay. B. Intravenous ψmRNA is expressedin spleen. Lipofectin-complexed ψmRNA (0.3 μg capTEVlucAn/mouse) wasadministered by tail vein injection. Animals were sacrificed at 2 and 4h post-injection and luciferase activities measured in aliquots (1/10th) of organs homogenized in lysis buffer. Values representluciferase activities in the whole organs. Expression of renillafollowing i.v. injection of mRNA into mouse tail vein. Data from twoindependently performed experiments are depicted in the left and rightpanels. Spleens were harvested and homogenized, and renilla activity wasmeasured in aliquots of the lysates. C. ψmRNA exhibits greater stabilityand translation in vivo. Lipofectin-complexed capTEVlucAn (0.3 μg/60μl/animal) with or without ψ modifications was delivered i.v. to mice.Animals were sacrificed at 1, 4 and 24 h post-injection, and ½ of theirspleens were processed for luciferase enzyme measurements (left panel)and the other half for RNA analyses (right panel). Luciferase activitieswere measured in aliquots (⅕th) of the homogenate made from half of thespleens. Plotted values represent luciferase activities in the wholespleen and are expressed as the mean±s.e.m. (n=3 or 4/point). D.Expression of firefly luciferase following intratracheal injection ofmRNA. capTEVluc-Y: capped, firefly luciferase-encodingpseudouridine-modified RNA. CapTEVluc-C: capped RNA with no nucleosidemodification.

FIG. 13. Protein production is dependent on the amount of mRNA deliveredintravenously in mice. The indicated amounts of lipofectin-complexednucleic acids, capTEVlucAn mRNA with or without ψ constituents andpCMVluc plasmid DNA in a volume of 60 μl/animal were delivered by i.v.injection into mice. Animals injected with mRNA or plasmid DNA weresacrificed at 6 h or 24 h post-injection, respectively, and luciferaseactivities were measured in aliquots ( 1/10th) of their spleenshomogenized in lysis buffer. The value from each animal is shown, andshort horizontal lines indicate the mean; N.D., not detectable.

FIG. 14. Expression of firefly luciferase following intratrachealdelivery of encoding mRNA. mRNA were complexed to lipofectin (or PEI, asnoted) and animals were injected with 0.3 μg firefly luciferase-encodingmRNA with or without ψ modification, then sacrificed 3 hours later.Lungs were harvested and homogenized, and luciferase activity wasmeasured in aliquots of the lysed organs.

FIG. 15. ψmRNA does not induce inflammatory mediators after pulmonarydelivery. Induction of TNF-α and IFN-α in serum following intratrachealdelivery of luciferase-encoding mRNA or ψmRNA. Serum levels of TNF-α andIFN-α were determined by ELISA 24 hours after mRNA delivery.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides RNA, oligoribonucleotide, and polyribonucleotidemolecules comprising pseudouridine or a modified nucleoside, genetherapy vectors comprising same, gene therapy methods and genetranscription silencing methods comprising same, methods of reducing animmunogenicity of same, and methods of synthesizing same.

In one embodiment, the present invention provides a messenger RNAcomprising a pseudouridine residue. In another embodiment, the messengerRNA encodes a protein of interest. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the present invention provides an RNA moleculeencoding a protein of interest, said RNA molecule comprising apseudouridine residue.

In another embodiment, the present invention provides invitro-transcribed RNA molecule, comprising a pseudouridine.

In another embodiment, the present invention provides an invitro-transcribed RNA molecule, comprising a modified nucleoside.

As provided herein, the present invention provides methods forsynthesizing in vitro-transcribed RNA molecules, comprisingpseudouridine and/or modified nucleosides.

In another embodiment, the present invention provides a messenger RNAmolecule comprising a pseudouridine residue

In another embodiment, an in vitro-transcribed RNA molecule of methodsand compositions of the present invention is synthesized by T7 phage RNApolymerase. In another embodiment, the molecule is synthesized by SP6phage RNA polymerase. In another embodiment, the molecule is synthesizedby T3 phage RNA polymerase. In another embodiment, the molecule issynthesized by a polymerase selected from the above polymerases.

In another embodiment, the in vitro-transcribed RNA molecule is anoligoribonucleotide. In another embodiment, the in vitro-transcribed RNAmolecule is a polyribonucleotide. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides an invitro-synthesized oligoribonucleotide, comprising a pseudouridine or amodified nucleoside, wherein the modified nucleoside is m⁵C, m⁵U, m⁶A,s²U, Ψ, or 2′-O-methyl-U.

In another embodiment, the present invention provides an invitro-synthesized polyribonucleotide, comprising a pseudouridine or amodified nucleoside, wherein the modified nucleoside is m⁵C, m⁵U, m⁶A,s²U, Ψ, or 2′-O-methyl-U.

In another embodiment, the in vitro-synthesized oligoribonucleotide orpolyribonucleotide is a short hairpin (sh)RNA. In another embodiment,the in vitro-synthesized oligoribonucleotide is a small interfering RNA(siRNA). In another embodiment, the in vitro-synthesizedoligoribonucleotide is any other type of oligoribonucleotide known inthe art. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, an RNA, oligoribonucleotide, orpolyribonucleotide molecule of methods and compositions of the presentinvention further comprises an open reading frame that encodes afunctional protein. In another embodiment, the RNA molecule oroligoribonucleotide molecule functions without encoding a functionalprotein (e.g. in transcriptional silencing), as an RNzyme, etc. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the RNA, oligoribonucleotide, orpolyribonucleotide molecule further comprises a poly-A tail. In anotherembodiment, the RNA, oligoribonucleotide, or polyribonucleotide moleculedoes not comprise a poly-A tail. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the RNA, oligoribonucleotide, orpolyribonucleotide molecule further comprises an m7GpppG cap. In anotherembodiment, the RNA, oligoribonucleotide, or polyribonucleotide moleculedoes not comprise an m7GpppG cap. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the RNA, oligoribonucleotide, orpolyribonucleotide molecule further comprises a cap-independenttranslational enhancer. In another embodiment, the RNA,oligoribonucleotide, or polyribonucleotide molecule does not comprise acap-independent translational enhancer. In another embodiment, thecap-independent translational enhancer is a tobacco etch virus (TEV)cap-independent translational enhancer. In another embodiment, thecap-independent translational enhancer is any other cap-independenttranslational enhancer known in the art. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the present invention provides a gene-therapyvector, comprising an in vitro-synthesized polyribonucleotide molecule,wherein the polyribonucleotide molecule comprises a pseudouridine or amodified nucleoside.

In another embodiment, an RNA, oligoribonucleotide, orpolyribonucleotide molecule of methods and compositions of the presentinvention comprises a pseudouridine. In another embodiment, the RNAmolecule or oligoribonucleotide molecule comprises a modifiednucleoside. In another embodiment, the RNA molecule oroligoribonucleotide molecule is an in vitro-synthesized RNA molecule oroligoribonucleotide. Each possibility represents a separate embodimentof the present invention.

“Pseudouridine” refers, in another embodiment, to m¹acp³Ψ(1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine. In anotherembodiment, the term refers to m¹Ψ (1-methylpseudouridine). In anotherembodiment, the term refers to Ψm (2′-O-methylpseudouridine. In anotherembodiment, the term refers to m⁵D (5-methyldihydrouridine). In anotherembodiment, the term refers to m³Ψ (3-methylpseudouridine). In anotherembodiment, the term refers to a pseudouridine moiety that is notfurther modified. In another embodiment, the term refers to amonophosphate, diphosphate, or triphosphate of any of the abovepseudouridines. In another embodiment, the term refers to any otherpseudouridine known in the art. Each possibility represents a separateembodiment of the present invention.

In another embodiment, an RNA, oligoribonucleotide, orpolyribonucleotide molecule of methods and compositions of the presentinvention is a therapeutic oligoribonucleotide.

In another embodiment, the present invention provides a method fordelivering a recombinant protein to a subject, the method comprising thestep of contacting the subject with an RNA, oligoribonucleotide,polyribonucleotide molecule, or a gene-therapy vector of the presentinvention, thereby delivering a recombinant protein to a subject.

In another embodiment, the present invention provides a double-strandedRNA (dsRNA) molecule comprising a pseudouridine or a modified nucleosideand further comprising an siRNA or short hairpin RNA (shRNA). In anotherembodiment, the dsRNA molecule is greater than 50 nucleotides in length.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the pseudouridine or a modified nucleoside iswithin the siRNA sequence. In another embodiment, the pseudouridine or amodified nucleoside is outside the siRNA sequence. In anotherembodiment, 1 or more pseudouridine and/or a modified nucleosideresidues are present both within and outside the siRNA sequence. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the siRNA or shRNA is contained internally in thedsRNA molecule. In another embodiment, the siRNA or shRNA is containedon one end of the dsRNA molecule. In another embodiment, one or moresiRNA or shRNA is contained on one end of the dsRNA molecule, whileanother one or more is contained internally. Each possibility representsa separate embodiment of the present invention.

In another embodiment, the length of an RNA, oligoribonucleotide, orpolyribonucleotide molecule (e.g. a single-stranded RNA (ssRNA) or dsRNAmolecule) of methods and compositions of the present invention isgreater than 30 nucleotides in length. In another embodiment, the RNAmolecule or oligoribonucleotide is greater than 35 nucleotides inlength. In another embodiment, the length is at least 40 nucleotides. Inanother embodiment, the length is at least 45 nucleotides. In anotherembodiment, the length is at least 55 nucleotides. In anotherembodiment, the length is at least 60 nucleotides. In anotherembodiment, the length is at least 60 nucleotides. In anotherembodiment, the length is at least 80 nucleotides. In anotherembodiment, the length is at least 90 nucleotides. In anotherembodiment, the length is at least 100 nucleotides. In anotherembodiment, the length is at least 120 nucleotides. In anotherembodiment, the length is at least 140 nucleotides. In anotherembodiment, the length is at least 160 nucleotides. In anotherembodiment, the length is at least 180 nucleotides. In anotherembodiment, the length is at least 200 nucleotides. In anotherembodiment, the length is at least 250 nucleotides. In anotherembodiment, the length is at least 300 nucleotides. In anotherembodiment, the length is at least 350 nucleotides. In anotherembodiment, the length is at least 400 nucleotides. In anotherembodiment, the length is at least 450 nucleotides. In anotherembodiment, the length is at least 500 nucleotides. In anotherembodiment, the length is at least 600 nucleotides. In anotherembodiment, the length is at least 700 nucleotides. In anotherembodiment, the length is at least 800 nucleotides. In anotherembodiment, the length is at least 900 nucleotides. In anotherembodiment, the length is at least 1000 nucleotides. In anotherembodiment, the length is at least 1100 nucleotides. In anotherembodiment, the length is at least 1200 nucleotides. In anotherembodiment, the length is at least 1300 nucleotides. In anotherembodiment, the length is at least 1400 nucleotides. In anotherembodiment, the length is at least 1500 nucleotides. In anotherembodiment, the length is at least 1600 nucleotides. In anotherembodiment, the length is at least 1800 nucleotides. In anotherembodiment, the length is at least 2000 nucleotides. In anotherembodiment, the length is at least 2500 nucleotides. In anotherembodiment, the length is at least 3000 nucleotides. In anotherembodiment, the length is at least 4000 nucleotides. In anotherembodiment, the length is at least 5000 nucleotides. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, a dsRNA molecule of methods and compositions ofthe present invention is manufactured by in vitro-transcription.

In another embodiment, the step of in vitro-transcription utilizes T7phage RNA polymerase. In another embodiment, the in vitro-transcriptionutilizes SP6 phage RNA polymerase. In another embodiment, the invitro-transcription utilizes T3 phage RNA polymerase. In anotherembodiment, the in vitro-transcription utilizes an RNA polymeraseselected from the above polymerases. In another embodiment, the invitro-transcription utilizes any other RNA polymerase known in the art.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the dsRNA molecule is capable of being processedby a cellular enzyme to yield the siRNA or shRNA. In another embodiment,the cellular enzyme is an endonuclease. In another embodiment, thecellular enzyme is Dicer. Dicer is an RNase III-family nuclease thatinitiates RNA interference (RNAi) and related phenomena by generation ofthe small RNAs that determine the specificity of these gene silencingpathways (Bernstein E, Caudy A A et al, Role for a bidentateribonuclease in the initiation step of RNA interference. Nature 2001;409(6818): 363-6). In another embodiment, the cellular enzyme is anyother cellular enzyme known in the art that is capable of cleaving adsRNA molecule. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the dsRNA molecule contains two siRNA or shRNA.In another embodiment, the dsRNA molecule contains three siRNA or shRNA.dsRNA molecule contains more than three siRNA or shRNA. In anotherembodiment, the siRNA and/or shRNA are liberated from the dsRNA moleculeby a cellular enzyme. Each possibility represents a separate embodimentof the present invention.

In another embodiment, the present invention provides a method foradministering an siRNA or shRNA to a cell, comprising administering adsRNA molecule of the present invention, wherein the cell processes thedsRNA molecule to yield the siRNA or shRNA, thereby administering asiRNA or shRNA to a cell.

In another embodiment, the nucleoside that is modified in an RNA,oligoribonucleotide, or polyribonucleotide molecule of methods andcompositions of the present invention is uridine (U). In anotherembodiment, the modified nucleoside is cytidine (C). In anotherembodiment, the modified nucleoside is adenine (A). In anotherembodiment the modified nucleoside is guanine (G). Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the modified nucleoside of methods andcompositions of the present invention is m⁵C (5-methylcytidine). Inanother embodiment, the modified nucleoside is m⁵U (5-methyluridine). Inanother embodiment, the modified nucleoside is m⁶A (N⁶-methyladenosine).In another embodiment, the modified nucleoside is s²U (2-thiouridine).In another embodiment, the modified nucleoside is Ψ (pseudouridine). Inanother embodiment, the modified nucleoside is Um (2′-O-methyluridine).

In other embodiments, the modified nucleoside is m¹A(1-methyladenosine); m²A (2-methyladenosine); Am (2′-O-methyladenosine);ms²m⁶A (2-methylthio-N⁶-methyladenosine); i⁶(N⁶-isopentenyladenosine);ms²i6A (2-methylthio-N⁶isopentenyladenosine); io⁶A(N⁶-(cis-hydroxyisopentenyl)adenosine); ms²io⁶A(2-methylthio-N⁶-(cis-hydroxyisopentenyl)adenosine); g⁶A(N⁶-glycinylcarbamoyladenosine); t⁶A (N⁶-threonylcarbamoyladenosine);ms²t⁶A (2-methylthio-N⁶-threonyl carbamoyladenosine); m⁶t⁶A(N⁶-methyl-N⁶-threonylcarbamoyladenosine); hn⁶A(N⁶-hydroxynorvalylcarbamoyladenosine); ms²hn⁶A(2-methylthio-N⁶-hydroxynorvalyl carbamoyladenosine); Ar(p)(2′-O-ribosyladenosine (phosphate)); I (inosine); m¹I (1-methylinosine);m¹Im (1,2′-O-dimethylinosine); m³C (3-methylcytidine); Cm(2′-O-methylcytidine); s²C (2-thiocytidine); ac⁴C(N⁴-acetylcytidine);f⁵C (5-formylcytidine); m⁵Cm (5,2′-O-dimethylcytidine); ac⁴Cm(N⁴-acetyl-2′-O-methylcytidine); k²C (lysidine); m¹G(1-methylguanosine); m²G (N²-methylguanosine); m⁷G (7-methylguanosine);Gm (2′-O-methylguanosine); m² ₂G (N²,N²-dimethylguanosine); m²Gm(N²,2′-O-dimethylguanosine); m² ₂Gm (N²,N²,2′-O-trimethylguanosine);Gr(p) (2′-O-ribosylguanosine (phosphate)); yW (wybutosine); o₂yW(peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodifiedhydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine);oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ(mannosyl-queuosine); preQ₀ (7-cyano-7-deazaguanosine); preQ₁(7-aminomethyl-7-deazaguanosine); G⁺ (archaeosine); D (dihydrouridine);m⁵Um (5,2′-O-dimethyluridine); s⁴U (4-thiouridine); m⁵s²U(5-methyl-2-thiouridine); s²Um (2-thio-2′-O-methyluridine); acp³U(3-(3-amino-3-carboxypropyl)uridine); ho⁵U (5-hydroxyuridine); mo⁵U(5-methoxyuridine); cmo⁵U (uridine 5-oxyacetic acid); mcmo⁵U (uridine5-oxyacetic acid methyl ester); chm⁵U(5-(carboxyhydroxymethyl)uridine)); mchm⁵U(5-(carboxyhydroxymethyl)uridine methyl ester); mcm⁵U(5-methoxycarbonylmethyluridine); mcm⁵Um(5-methoxycarbonylmethyl-2′-O-methyluridine); mcm⁵s²U(5-methoxycarbonylmethyl-2-thiouridine); nm⁵S²U(5-aminomethyl-2-thiouridine); mnm⁵U (5-methylaminomethyluridine);mnm⁵s²U (5-methylaminomethyl-2-thiouridine); mnm⁵se²U(5-methylaminomethyl-2-selenouridine); ncm⁵U (5-carbamoylmethyluridine);ncm⁵Um (5-carbamoylmethyl-2′-O-methyluridine); cmnm⁵U(5-carboxymethylaminomethyluridine); cmnm⁵Um(5-carboxymethylaminomethyl-2′-O-methyluridine); cmnm⁵s²U(5-carboxymethylaminomethyl-2-thiouridine); m⁶ ₂A(N⁶,N⁶-dimethyladenosine); Im (2′-O-methylinosine);m⁴C(N⁴-methylcytidine); m⁴Cm (N⁴,2′-O-dimethylcytidine); hm⁵C(5-hydroxymethylcytidine); m³U (3-methyluridine); cm⁵U(5-carboxymethyluridine); m⁶Am (N⁶,2′-O-dimethyladenosine); m⁶ ₂Am(N⁶,N⁶,O-2′-trimethyladenosine); m^(2,7)G (N²,7-dimethylguanosine);m2′2′7G (N²,N²,7-trimethylguanosine); m³Um (3,2′-O-dimethyluridine); m⁵D(5-methyldihydrouridine); f⁵ Cm (5-formyl-2′-O-methylcytidine); m¹Gm(1,2′-O-dimethylguanosine); m¹Am (1,2′-O-dimethyladenosine); τm⁵U(5-taurinomethyluridine); τm⁵s²U (5-taurinomethyl-2-thiouridine));imG-14 (4-demethylwyosine); imG2 (isowyosine); or ac⁶A(N⁶-acetyladenosine). Each possibility represents a separate embodimentof the present invention.

In another embodiment, an RNA, oligoribonucleotide, orpolyribonucleotide molecule of methods and compositions of the presentinvention comprises a combination of 2 or more of the abovemodifications. In another embodiment, the RNA molecule oroligoribonucleotide molecule comprises a combination of 3 or more of theabove modifications. In another embodiment, the RNA molecule oroligoribonucleotide molecule comprises a combination of more than 3 ofthe above modifications. Each possibility represents a separateembodiment of the present invention.

In another embodiment, between 0.1% and 100% of the residues in the RNA,oligoribonucleotide, or polyribonucleotide molecule of methods andcompositions of the present invention are modified (e.g. either by thepresence of pseudouridine or a modified nucleoside base). In anotherembodiment, 0.1% of the residues are modified. In another embodiment,0.2%. In another embodiment, the fraction is 0.3%. In anotherembodiment, the fraction is 0.4%. In another embodiment, the fraction is0.5%. In another embodiment, the fraction is 0.6%. In anotherembodiment, the fraction is 0.8%. In another embodiment, the fraction is1%. In another embodiment, the fraction is 1.5%. In another embodiment,the fraction is 2%. In another embodiment, the fraction is 2.5%. Inanother embodiment, the fraction is 3%. In another embodiment, thefraction is 4%. In another embodiment, the fraction is 5%. In anotherembodiment, the fraction is 6%. In another embodiment, the fraction is8%. In another embodiment, the fraction is 10%. In another embodiment,the fraction is 12%. In another embodiment, the fraction is 14%. Inanother embodiment, the fraction is 16%. In another embodiment, thefraction is 18%. In another embodiment, the fraction is 20%. In anotherembodiment, the fraction is 25%. In another embodiment, the fraction is30%. In another embodiment, the fraction is 35%. In another embodiment,the fraction is 40%. In another embodiment, the fraction is 45%. Inanother embodiment, the fraction is 50%. In another embodiment, thefraction is 60%. In another embodiment, the fraction is 70%. In anotherembodiment, the fraction is 80%. In another embodiment, the fraction is90%. In another embodiment, the fraction is 100%.

In another embodiment, the fraction is less than 5%. In anotherembodiment, the fraction is less than 3%. In another embodiment, thefraction is less than 1%. In another embodiment, the fraction is lessthan 2%. In another embodiment, the fraction is less than 4%. In anotherembodiment, the fraction is less than 6%. In another embodiment, thefraction is less than 8%. In another embodiment, the fraction is lessthan 10%. In another embodiment, the fraction is less than 12%. Inanother embodiment, the fraction is less than 15%. In anotherembodiment, the fraction is less than 20%. In another embodiment, thefraction is less than 30%. In another embodiment, the fraction is lessthan 40%. In another embodiment, the fraction is less than 50%. Inanother embodiment, the fraction is less than 60%. In anotherembodiment, the fraction is less than 70%.

In another embodiment, 0.1% of the residues of a given nucleotide(uridine, cytidine, guanosine, or adenine) are modified. In anotherembodiment, the fraction of the nucleotide is 0.2%. In anotherembodiment, the fraction is 0.3%. In another embodiment, the fraction is0.4%. In another embodiment, the fraction is 0.5%. In anotherembodiment, the fraction is 0.6%. In another embodiment, the fraction is0.8%. In another embodiment, the fraction is 1%. In another embodiment,the fraction is 1.5%. In another embodiment, the fraction is 2%. Inanother embodiment, the fraction is 2.5%. In another embodiment, thefraction is 3%. In another embodiment, the fraction is 4%. In anotherembodiment, the fraction is 5%. In another embodiment, the fraction is6%. In another embodiment, the fraction is 8%. In another embodiment,the fraction is 10%. In another embodiment, the fraction is 12%. Inanother embodiment, the fraction is 14%. In another embodiment, thefraction is 16%. In another embodiment, the fraction is 18%. In anotherembodiment, the fraction is 20%. In another embodiment, the fraction is25%. In another embodiment, the fraction is 30%. In another embodiment,the fraction is 35%. In another embodiment, the fraction is 40%. Inanother embodiment, the fraction is 45%. In another embodiment, thefraction is 50%. In another embodiment, the fraction is 60%. In anotherembodiment, the fraction is 70%. In another embodiment, the fraction is80%. In another embodiment, the fraction is 90%. In another embodiment,the fraction is 100%.

In another embodiment, the fraction of the given nucleotide is less than8%. In another embodiment, the fraction is less than 10%. In anotherembodiment, the fraction is less than 5%. In another embodiment, thefraction is less than 3%. In another embodiment, the fraction is lessthan 1%. In another embodiment, the fraction is less than 2%. In anotherembodiment, the fraction is less than 4%. In another embodiment, thefraction is less than 6%. In another embodiment, the fraction is lessthan 12%. In another embodiment, the fraction is less than 15%. Inanother embodiment, the fraction is less than 20%. In anotherembodiment, the fraction is less than 30%. In another embodiment, thefraction is less than 40%. In another embodiment, the fraction is lessthan 50%. In another embodiment, the fraction is less than 60%. Inanother embodiment, the fraction is less than 70%.

In another embodiment, the terms “ribonucleotide,”“oligoribonucleotide,” and “polyribonucleotide” refers to a string of atleast 2 base-sugar-phosphate combinations. The term includes, in anotherembodiment, compounds comprising nucleotides in which the sugar moietyis ribose. In another embodiment, the term includes both RNA and RNAderivates in which the backbone is modified. “Nucleotides” refers, inanother embodiment, to the monomeric units of nucleic acid polymers. RNAmay be, in an other embodiment, in the form of a tRNA (transfer RNA),snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA),anti-sense RNA, small inhibitory RNA (siRNA), micro RNA (miRNA) andribozymes. The use of siRNA and miRNA has been described (Caudy A A etal, Genes & Devel 16: 2491-96 and references cited therein). Inaddition, these forms of RNA may be single, double, triple, or quadruplestranded. The term also includes, in another embodiment, artificialnucleic acids that may contain other types of backbones but the samebases. In another embodiment, the artificial nucleic acid is a PNA(peptide nucleic acid). PNA contain peptide backbones and nucleotidebases and are able to bind, in another embodiment, to both DNA and RNAmolecules. In another embodiment, the nucleotide is oxetane modified. Inanother embodiment, the nucleotide is modified by replacement of one ormore phosphodiester bonds with a phosphorothioate bond. In anotherembodiment, the artificial nucleic acid contains any other variant ofthe phosphate backbone of native nucleic acids known in the art. The useof phosphothiorate nucleic acids and PNA are known to those skilled inthe art, and are described in, for example, Neilsen P E, Curr OpinStruct Biol 9:353-57; and Raz N K et al Biochem Biophys Res Commun.297:1075-84. The production and use of nucleic acids is known to thoseskilled in art and is described, for example, in Molecular Cloning,(2001), Sambrook and Russell, eds. and Methods in Enzymology: Methodsfor molecular cloning in eukaryotic cells (2003) Purchio and G. C.Fareed. Each nucleic acid derivative represents a separate embodiment ofthe present invention

In another embodiment, the term “oligoribonucleotide” refers to a stringcomprising fewer than 25 nucleotides (nt). In another embodiment,“oligoribonucleotide” refers to a string of fewer than 24 nucleotides.In another embodiment, “oligoribonucleotide” refers to a string of fewerthan 23 nucleotides. In another embodiment, “oligoribonucleotide” refersto a string of fewer than 22 nucleotides. In another embodiment,“oligoribonucleotide” refers to a string of fewer than 21 nucleotides.In another embodiment, “oligoribonucleotide” refers to a string of fewerthan 20 nucleotides. In another embodiment, “oligoribonucleotide” refersto a string of fewer than 19 nucleotides. In another embodiment,“oligoribonucleotide” refers to a string of fewer than 18 nucleotides.In another embodiment, “oligoribonucleotide” refers to a string of fewerthan 17 nucleotides. In another embodiment, “oligoribonucleotide” refersto a string of fewer than 16 nucleotides. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the term “polyribonucleotide” refers to a stringcomprising more than 25 nucleotides (nt). In another embodiment,“polyribonucleotide” refers to a string of more than 26 nucleotides. Inanother embodiment, “polyribonucleotide” refers to a string of more than28 nucleotides. In another embodiment, “the term” refers to a string ofmore than 30 nucleotides. In another embodiment, “the term” refers to astring of more than 32 nucleotides. In another embodiment, “the term”refers to a string of more than 35 nucleotides. In another embodiment,“the term” refers to a string of more than 40 nucleotides. In anotherembodiment, “the term” refers to a string of more than 50 nucleotides.In another embodiment, “the term” refers to a string of more than 60nucleotides. In another embodiment, “the term” refers to a string ofmore than 80 nucleotides. In another embodiment, “the term” refers to astring of more than 100 nucleotides. In another embodiment, “the term”refers to a string of more than 120 nucleotides. In another embodiment,“the term” refers to a string of more than 150 nucleotides. In anotherembodiment, “the term” refers to a string of more than 200 nucleotides.In another embodiment, “the term” refers to a string of more than 300nucleotides. In another embodiment, “the term” refers to a string ofmore than 400 nucleotides. In another embodiment, “the term” refers to astring of more than 500 nucleotides. In another embodiment, “the term”refers to a string of more than 600 nucleotides. In another embodiment,“the term” refers to a string of more than 800 nucleotides. In anotherembodiment, “the term” refers to a string of more than 1000 nucleotides.In another embodiment, “the term” refers to a string of more than 1200nucleotides. In another embodiment, “the term” refers to a string ofmore than 1400 nucleotides. In another embodiment, “the term” refers toa string of more than 1600 nucleotides. In another embodiment, “theterm” refers to a string of more than 1800 nucleotides. In anotherembodiment, “the term” refers to a string of more than 2000 nucleotides.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the present invention provides a method forinducing a mammalian cell to produce a protein of interest, comprisingcontacting the mammalian cell with an in vitro-synthesized RNA moleculeencoding the recombinant protein, the in vitro-synthesized RNA moleculecomprising a pseudouridine or a modified nucleoside, thereby inducing amammalian cell to produce a protein of interest. In another embodiment,the protein of interest is a recombinant protein. Each possibilityrepresents a separate embodiment of the present invention.

“Encoding” refers, in another embodiment, to an RNA molecule thatcontains a gene that encodes the protein of interest. In anotherembodiment, the RNA molecule comprises an open reading frame thatencodes the protein of interest. In another embodiment, 1 or more otherproteins is also encoded. In another embodiment, the protein of interestis the only protein encoded. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides a method ofinducing a mammalian cell to produce a recombinant protein, comprisingcontacting the mammalian cell with an in vitro-transcribed RNA moleculeencoding the recombinant protein, the in vitro-transcribed RNA moleculefurther comprising a pseudouridine or a modified nucleoside, therebyinducing a mammalian cell to produce a recombinant protein.

In another embodiment, an RNA, oligoribonucleotide, orpolyribonucleotide molecule of methods and compositions of the presentinvention is translated in the cell more efficiently than an unmodifiedRNA molecule with the same sequence. In another embodiment, the RNA,oligoribonucleotide, or polyribonucleotide molecule exhibits enhancedability to be translated by a target cell. In another embodiment,translation is enhanced by a factor of 2-fold relative to its unmodifiedcounterpart. In another embodiment, translation is enhanced by a 3-foldfactor. In another embodiment, translation is enhanced by a 5-foldfactor. In another embodiment, translation is enhanced by a 7-foldfactor. In another embodiment, translation is enhanced by a 10-foldfactor. In another embodiment, translation is enhanced by a 15-foldfactor. In another embodiment, translation is enhanced by a 20-foldfactor. In another embodiment, translation is enhanced by a 50-foldfactor. In another embodiment, translation is enhanced by a 100-foldfactor. In another embodiment, translation is enhanced by a 200-foldfactor. In another embodiment, translation is enhanced by a 500-foldfactor. In another embodiment, translation is enhanced by a 1000-foldfactor. In another embodiment, translation is enhanced by a 2000-foldfactor. In another embodiment, the factor is 10-1000-fold. In anotherembodiment, the factor is 10-100-fold. In another embodiment, the factoris 10-200-fold. In another embodiment, the factor is 10-300-fold. Inanother embodiment, the factor is 10-500-fold. In another embodiment,the factor is 20-1000-fold. In another embodiment, the factor is30-1000-fold. In another embodiment, the factor is 50-1000-fold. Inanother embodiment, the factor is 100-1000-fold. In another embodiment,the factor is 200-1000-fold. In another embodiment, translation isenhanced by any other significant amount or range of amounts. Eachpossibility represents a separate embodiment of the present invention.

Methods of determining translation efficiency are well known in the art,and include, e.g. measuring the activity of an encoded reporter protein(e.g luciferase or renilla [Examples herein] or green fluorescentprotein [Wall A A, Phillips A M et al, Effective translation of thesecond cistron in two Drosophila dicistronic transcripts is determinedby the absence of in-frame AUG codons in the first cistron. J Biol Chem2005; 280(30): 27670-8]), or measuring radioactive label incorporatedinto the translated protein (Ngosuwan J, Wang N M et al, Roles ofcytosolic Hsp70 and Hsp40 molecular chaperones in post-translationaltranslocation of presecretory proteins into the endoplasmic reticulum. JBiol Chem 2003; 278(9): 7034-42). Each method represents a separateembodiment of the present invention.

In expression studies provided herein, translation was measured from RNAcomplexed to Lipofectin® (Gibco BRL, Gaithersburg, Md., USA) andinjected into the tail vein of mice. In the spleen lysates,pseudouridine-modified RNA was translated significantly more efficientlythan unmodified RNA (FIG. 12B). Under the conditions utilized herein,efficiency of transfection-based methods of the present inventioncorrelates with the ability of the transfection reagent to penetrateinto tissues, providing an explanation for why the effect was mostpronounced in spleen cells. Splenic blood flow is an open system, withblood contents directly contacting red and white pulp elements includinglymphoid cells.

In another experiment, in vitro phosphorylation assays were performedusing recombinant human PKR and its substrate, eIF2α in the presence ofcapped, renilla-encoding mRNA (0.5 and 0.05 ng/μl). mRNA containingpseudouridine (Ψ) did not activate PKR, as detected by lack of bothself-phosphorylation of PKR and phosphorylation of eIF2α, while RNAwithout nucleoside modification and mRNA with m5C modification activatedPKR. Phosphorylated eIF2α is known to block initiation of mRNAtranslation, therefore lack of phosphorylation enables, in anotherembodiment, enhanced translation of the mRNA containing pseudouridine(Ψ).

In another embodiment, the enhanced translation is in a cell (relativeto translation in the same cell of an unmodified RNA molecule with thesame sequence; Examples 10-11). In another embodiment, the enhancedtranslation is in vitro (e.g. in an in vitro translation mix or areticulocyte lysate; Examples 10-11. In another embodiment, the enhancedtranslation is in vivo (Example 13). In each case, the enhancedtranslation is relative to an unmodified RNA molecule with the samesequence, under the same conditions. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the RNA, oligoribonucleotide, orpolyribonucleotide molecule of methods and compositions of the presentinvention is significantly less immunogenic than an unmodified invitro-synthesized RNA molecule with the same sequence. In anotherembodiment, the modified RNA molecule is 2-fold less immunogenic thanits unmodified counterpart. In another embodiment, immunogenicity isreduced by a 3-fold factor. In another embodiment, immunogenicity isreduced by a 5-fold factor. In another embodiment, immunogenicity isreduced by a 7-fold factor. In another embodiment, immunogenicity isreduced by a 10-fold factor. In another embodiment, immunogenicity isreduced by a 15-fold factor. In another embodiment, immunogenicity isreduced by a 20-fold factor. In another embodiment, immunogenicity isreduced by a 50-fold factor. In another embodiment, immunogenicity isreduced by a 100-fold factor. In another embodiment, immunogenicity isreduced by a 200-fold factor. In another embodiment, immunogenicity isreduced by a 500-fold factor. In another embodiment, immunogenicity isreduced by a 1000-fold factor. In another embodiment, immunogenicity isreduced by a 2000-fold factor. In another embodiment, immunogenicity isreduced by another fold difference.

In another embodiment, “significantly less immunogenic” refers to adetectable decrease in immunogenicity. In another embodiment, the termrefers to a fold decrease in immunogenicity (e.g. 1 of the folddecreases enumerated above). In another embodiment, the term refers to adecrease such that an effective amount of the RNA, oligoribonucleotide,or polyribonucleotide molecule can be administered without triggering adetectable immune response. In another embodiment, the term refers to adecrease such that the RNA, oligoribonucleotide, or polyribonucleotidemolecule can be repeatedly administered without eliciting an immuneresponse sufficient to detectably reduce expression of the recombinantprotein. In another embodiment, the decrease is such that the RNA,oligoribonucleotide, or polyribonucleotide molecule can be repeatedlyadministered without eliciting an immune response sufficient toeliminate detectable expression of the recombinant protein.

“Effective amount” of the RNA, oligoribonucleotide, orpolyribonucleotide molecule refers, in another embodiment, to an amountsufficient to exert a therapeutic effect. In another embodiment, theterm refers to an amount sufficient to elicit expression of a detectableamount of the recombinant protein. Each possibility represents aseparate embodiment of the present invention.

Reduced immunogenicity of RNA, oligoribonucleotide, andpolyribonucleotide molecules of the present invention is demonstratedherein (Examples 1-8).

Methods of determining immunogenicity are well known in the art, andinclude, e.g. measuring secretion of cytokines (e.g. IL-12, IFN-α,TNF-α, RANTES, MIP-1α or β, IL-6, IFN-β, or IL-8; Examples herein),measuring expression of DC activation markers (e.g. CD83, HLA-DR, CD80and CD86; Examples herein), or measuring ability to act as an adjuvantfor an adaptive immune response. Each method represents a separateembodiment of the present invention.

In another embodiment, the relative immunogenicity of the modifiednucleotide and its unmodified counterpart are determined by determiningthe quantity of the modified nucleotide required to elicit one of theabove responses to the same degree as a given quantity of the unmodifiednucleotide. For example, if twice as much modified nucleotide isrequired to elicit the same response, than the modified nucleotide istwo-fold less immunogenic than the unmodified nucleotide.

In another embodiment, the relative immunogenicity of the modifiednucleotide and its unmodified counterpart are determined by determiningthe quantity of cytokine (e.g. IL-12, IFN-α, TNF-α, RANTES, MIP-1α or β,IL-6, IFN-β, or IL-8) secreted in response to administration of themodified nucleotide, relative to the same quantity of the unmodifiednucleotide. For example, if one-half as much cytokine is secreted, thanthe modified nucleotide is two-fold less immunogenic than the unmodifiednucleotide. In another embodiment, background levels of stimulation aresubtracted before calculating the immunogenicity in the above methods.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, a method of present invention further comprisesmixing the RNA, oligoribonucleotide, or polyribonucleotide molecule witha transfection reagent prior to the step of contacting. In anotherembodiment, a method of present invention further comprisesadministering the RNA, oligoribonucleotide, or polyribonucleotidemolecule together with the transfection reagent. In another embodiment,the transfection reagent is a cationic lipid reagent (Example 3).

In another embodiment, the transfection reagent is a lipid-basedtransfection reagent. In another embodiment, the transfection reagent isa protein-based transfection reagent. In another embodiment, thetransfection reagent is a polyethyleneimine based transfection reagent.In another embodiment, the transfection reagent is calcium phosphate. Inanother embodiment, the transfection reagent is Lipofectin® orLipofectamine®. In another embodiment, the transfection reagent is anyother transfection reagent known in the art.

In another embodiment, the transfection reagent forms a liposome.Liposomes, in another embodiment, increase intracellular stability,increase uptake efficiency and improve biological activity. In anotherembodiment, liposomes are hollow spherical vesicles composed of lipidsarranged in a similar fashion as those lipids which make up the cellmembrane. They have, in another embodiment, an internal aqueous spacefor entrapping water soluble compounds and range in size from 0.05 toseveral microns in diameter. In another embodiment, liposomes candeliver RNA to cells in a biologically active form.

Each type of transfection reagent represents a separate embodiment ofthe present invention.

In another embodiment, the target cell of methods of the presentinvention is an antigen-presenting cell. In another embodiment, the cellis an animal cell. In another embodiment, the cell is a dendritic cell(Example 11). In another embodiment, the cell is a neural cell. Inanother embodiment, the cell is a brain cell (Example 13). In anotherembodiment, the cell is a spleen cell. In another embodiment, the cellis a lymphoid cell. In another embodiment, the cell is a lung cell(Example 13). In another embodiment, the cell is a skin cell. In anotherembodiment, the cell is a keratinocyte. In another embodiment, the cellis an endothelial cell. In another embodiment, the cell is an astrocyte,a microglial cell, or a neuron (Example 13). In another embodiment, thecell is an alveolar cell (Example 13). In another embodiment, the cellis a surface alveolar cell (Example 13). In another embodiment, the cellis an alveolar macrophage. In another embodiment, the cell is analveolar pneumocyte. In another embodiment, the cell is a vascularendothelial cell. In another embodiment, the cell is a mesenchymal cell.In another embodiment, the cell is an epithelial cell. In anotherembodiment, the cell is a hematopoietic cell. In another embodiment, thecell is colonic epithelium cell. In another embodiment, the cell is alung epithelium cell. In another embodiment, the cell is a bone marrowcell.

In other embodiments, the target cell is a Claudius' cell, Hensen cell,Merkel cell, Müller cell, Paneth cell, Purkinje cell, Schwann cell,Sertoli cell, acidophil cell, acinar cell, adipoblast, adipocyte, brownor white alpha cell, amacrine cell, beta cell, capsular cell,cementocyte, chief cell, chondroblast, chondrocyte, chromaffin cell,chromophobic cell, corticotroph, delta cell, Langerhans cell, folliculardendritic cell, enterochromaffin cell, ependymocyte, epithelial cell,basal cell, squamous cell, endothelial cell, transitional cell,erythroblast, erythrocyte, fibroblast, fibrocyte, follicular cell, germcell, gamete, ovum, spermatozoon, oocyte, primary oocyte, secondaryoocyte, spermatid, spermatocyte, primary spermatocyte, secondaryspermatocyte, germinal epithelium, giant cell, glial cell, astroblast,astrocyte, oligodendroblast, oligodendrocyte, glioblast, goblet cell,gonadotroph, granulosa cell, haemocytoblast, hair cell, hepatoblast,hepatocyte, hyalocyte, interstitial cell, juxtaglomerular cell,keratinocyte, keratocyte, lemmal cell, leukocyte, granulocyte, basophil,eosinophil, neutrophil, lymphoblast, B-lymphoblast, T-lymphoblast,lymphocyte, B-lymphocyte, T-lymphocyte, helper induced T-lymphocyte, Th1T-lymphocyte, Th2 T-lymphocyte, natural killer cell, thymocyte,macrophage, Kupffer cell, alveolar macrophage, foam cell, histiocyte,luteal cell, lymphocytic stem cell, lymphoid cell, lymphoid stem cell,macroglial cell, mammotroph, mast cell, medulloblast, megakaryoblast,megakaryocyte, melanoblast, melanocyte, mesangial cell, mesothelialcell, metamyelocyte, monoblast, monocyte, mucous neck cell, muscle cell,cardiac muscle cell, skeletal muscle cell, smooth muscle cell,myelocyte, myeloid cell, myeloid stem cell, myoblast, myoepithelialcell, myofibrobast, neuroblast, neuroepithelial cell, neuron,odontoblast, osteoblast, osteoclast, osteocyte, oxyntic cell,parafollicular cell, paraluteal cell, peptic cell, pericyte, peripheralblood mononuclear cell, phaeochromocyte, phalangeal cell, pinealocyte,pituicyte, plasma cell, platelet, podocyte, proerythroblast,promonocyte, promyeloblast, promyelocyte, pronormoblast, reticulocyte,retinal pigment epithelial cell, retinoblast, small cell, somatotroph,stem cell, sustentacular cell, teloglial cell, or zymogenic cell. Eachpossibility represents a separate embodiment of the present invention.

A variety of disorders may be treated by employing methods of thepresent invention including, inter alia, monogenic disorders, infectiousdiseases, acquired disorders, cancer, and the like. Exemplary monogenicdisorders include ADA deficiency, cystic fibrosis,familial-hypercholesterolemia, hemophilia, chronic ganulomatous disease,Duchenne muscular dystrophy, Fanconi anemia, sickle-cell anemia,Gaucher's disease, Hunter syndrome, X-linked SCID, and the like. Inanother embodiment, the disorder treated involves one of the proteinslisted below. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the recombinant protein encoded by an RNA,oligoribonucleotide, or polyribonucleotide molecule of methods andcompositions of the present invention is ecto-nucleoside triphosphatediphosphohydrolase.

In another embodiment, the recombinant protein is erythropoietin (EPO).

In other embodiments, the encoded recombinant protein is ABCA4; ABCD3;ACADM; AGL; AGT; ALDH4A1; ALPL; AMPD1; APOA2; AVSD1; BRCD2; C1QA; C1QB;C1QG; CBA; C8B; CACNA1S; CCV; CD3Z; CDC2L1; CHML; CHS1; CIAS1; CLCNKB;CMD1A; CMH2; CMM; COL11A1; COL8A2; COL9A2; CPT2; CRB1; CSE; CSF3R; CTPA;CTSK; DBT; DIO1; DISC1; DPYD; EKV; ENO1; ENO1P; EPB41; EPHX1; F13B; F5;FCGR2A; FCGR2B; FCGR3A; FCHL; FH; FMO3; FMO4; FUCA1; FY; GALE; GBA;GFND; GJA8; GJB3; GLC3B; HF1; HMGCL; HPC1; HRD; HRPT2; HSD3B2; HSPG2;KCNQ4; KCS; KIF1B; LAMB3; LAMC2; LGMD1B; LMNA; LOR; MCKD1; MCL1; MPZ;MTHFR; MTR; MUTYH; MYOC; NB; NCF2; NEM1; NPHS2; NPPA; NRAS; NTRK1;OPTA2; PBX1; PCHC; PGD; PHA2A; PHGDH; PKLR; PKP1; PLA2G2A; PLOD; PPDX;PPT1; PRCC; PRG4; PSEN2; PTOS1; REN; RFX5; RHD; RMD1; RPE65; SCCD;SERPINC1; SJS1; SLC19A2; SLC2A1; SPG23; SPTA1; TAL1; TNFSF6; TNNT2;TPM3; TSHB; UMPK; UOX; UROD; USH2A; VMGLOM; VWS; WS2B; ABCB11; ABCG5;ABCG8; ACADL; ACP1; AGXT; AHHR; ALMS1; ALPP; ALS2; APOB; BDE; BDMR; BJS;BMPR2; CHRNA1; CMCWTD; CNGA3; COL3A1; COL4A3; COL4A4; COL6A3; CPS1;CRYGA; CRYGEP1; CYP1B1; CYP27A1; DBI; DES; DYSF; EDAR; EFEMP1; EIF2AK3;ERCC3; FSHR; GINGF; GLC1B; GPD2; GYPC; HADHA; HADHB; HOXD13; HPE2; IGKC;IHH; IRS1; ITGA6; KHK; KYNU; LCT; LHCGR; LSFC; MSH2; MSH6; NEB; NMTC;NPHP1; PAFAH1P1; PAX3; PAX8; PMS1; PNKD; PPH1; PROC; REG1A; SAG; SFTPB;SLC11A1; SLC3A1; SOS1; SPG4; SRD5A2; TCL4; TGFA; TMD; TPO; UGT1A@; UV24;WSS; XDH; ZAP70; ZFHX1B; ACAA1; AGS1; AGTR1; AHSG; AMT; ARMET; BBS3;BCHE; BCPM; BTD; CASR; CCR2; CCR5; CDL1; CMT2B; COL7A1; CP; CPO; CRV;CTNNB1; DEM; ETM1; FANCD2; FIH; FOXL2; GBE1; GLB1; GLC1C; GNAI2; GNAT1;GP9; GPX1; HGD; HRG; ITIH1; KNG; LPP; LRS1; MCCC1; MDS1; MHS4; MITF;MLH1; MYL3; MYMY; OPA1; P2RY12; PBXP1; PCCB; POU1F1; PPARG; PRO51;PTHR1; RCA1; RHO; SCA7; SCLC1; SCN5A; SI; SLC25A20; SLC2A2; TF; TGFBR2;THPO; THRB; TKT; TM4SF1; TRH; UMPS; UQCRC1; USH3A; VHL; WS2A; XPC;ZNF35; ADH1B; ADH1C; AFP; AGA; AIH2; ALB; ASMD; BFHD; CNGA1; CRBM; DCK;DSPP; DTDP2; ELONG; ENAM; ETFDH; EVC; F11; FABP2; FGA; FGB; FGFR3; FGG;FSHMD1A; GC; GNPTA; GNRHR; GYPA; HCA; HCL2; HD; HTN3; HVBS6; IDUA; IF;JPD; KIT; KLKB1; LQT4; MANBA; MLLT2; MSX1; MTP; NR3C2; PBT; PDE6B; PEE1;PITX2; PKD2; QDPR; SGCB; SLC25A4; SNCA; SOD3; STATH; TAPVR1; TYS; WBS2;WFS1; WHCR; ADAMTS2; ADRB2; AMCN; AP3B1; APC; ARSB; B4GALT7; BHR1; C6;C7; CCAL2; CKN1; CMDJ; CRHBP; CSF1R; DHFR; DIAPH1; DTR; EOS; EPD; ERVR;F12; FBN2; GDNF; GHR; GLRA1; GM2A; HEXB; HSD17B4; ITGA2; KFS; LGMD1A;LOX; LTC4S; MAN2A1; MCC; MCCC2; MSH3; MSX2; NR3C1; PCSK1; PDE6A; PFBI;RASA1; SCZD1; SDHA; SGCD; SLC22A5; SLC26A2; SLC6A3; SM1; SMA@; SMN1;SMN2; SPINK5; TCOF1; TELAB1; TGFBI; ALDH5A1; ARG1; AS; ASSP2; BCKDHB;BF; C2; C4A; CDKN1A; COL10A1; COL11A2; CYP21A2; DYX2; EJM1; ELOVL4;EPM2A; ESR1; EYA4; F13A1; FANCE; GCLC; GJA1; GLYS1; GMPR; GSE; HCR; HFE;HLA-A; HLA-DPB1; HLA-DRA; HPFH; ICS1; IDDM1; IFNGR1; IGAD1; IGF2R; ISCW;LAMA2; LAP; LCA5; LPA; MCDR1; MOCS1; MUT; MYB; NEU1; NKS1; NYS2; OA3;ODDD; OFC1; PARK2; PBCA; PBCRA1; PDB1; PEX3; PEX6; PEX7; PKHD1; PLA2G7;PLG; POLH; PPAC; PSORS1; PUJO; RCD1; RDS; RHAG; RP14; RUNX2; RWS; SCA1;SCZD3; SIASD; SOD2; ST8; TAP1; TAP2; TFAP2B; TNDM; TNF; TPBG; TPMT;TULP1; WISP3; AASS; ABCB1; ABCB4; ACHE; AQP1; ASL; ASNS; AUTS1; BPGM;BRAF; C7orf2; CACNA2D1; CCM1; CD36; CFTR; CHORDOMA; CLCN1; CMH6; CMT2D;COL1A2; CRS; CYMD; DFNA5; DLD; DYT11; EEC1; ELN; ETV1; FKBP6; GCK;GHRHR; GHS; GLI3; GPDS1; GUSB; HLXB9; HOXA13; HPFH2; HRX; IAB; IMMP2L;KCNH2; LAMB1; LEP; MET; NCF1; NM; OGDH; OPN1SW; PEX1; PGAM2; PMS2; PON1;PPP1R3A; PRSS1; PTC; PTPN12; RP10; RP9; SERPINE1; SGCE; SHFM1; SHH;SLC26A3; SLC26A4; SLOS; SMAD1; TBXAS1; TWIST; ZWS1; ACHM3; ADRB3; ANK1;CA1; CA2; CCAL1; CLN8; CMT4A; CNGB3; COH1; CPP; CRH; CYP11B1; CYP11B2;DECR1; DPYS; DURST; EBS1; ECA1; EGI; EXT1; EYA1; FGFR1; GNRH1; GSR;GULOP; HR; KCNQ3; KFM; KWE; LGCR; LPL; MCPH1; MOS; MYC; NAT1; NAT2;NBS1; PLAT; PLEC1; PRKDC; PXMP3; RP1; SCZD6; SFTPC; SGM1; SPG5A; STAR;TG; TRPS1; TTPA; VMD1; WRN; ABCA1; ABL1; ABO; ADAMTS13; AK1; ALAD;ALDH1A1; ALDOB; AMBP; AMCD1; ASS; BDMF; BSCL; C5; CDKN2A; CHAC; CLA1;CMD1B; COL5A1; CRAT; DBH; DNAI1; DYS; DYT1; ENG; FANCC; FBP1; FCMD;FRDA; GALT; GLDC; GNE; GSM1; GSN; HSD17B3; HSN1; IBM2; INVS; JBTS1;LALL; LCCS1; LCCS; LGMD2H; LMX1B; MLLT3; MROS; MSSE; NOTCH1; ORM1;PAPPA; PIP5K1B; PTCH; PTGS1; RLN1; RLN2; RMRP; ROR2; RPD1; SARDH;SPTLC1; STOM; TDFA; TEK; TMC1; TRIM32; TSC1; TYRP1; XPA; CACNB2;COL17A1; CUBN; CXCL12; CYP17; CYP2C19; CYP2C9; EGR2; EMX2; ERCC6; FGFR2;HK1; HPS1; IL2RA; LGI1; L1PA; MAT1A; MBL2; MKI67; MXI1; NODAL; OAT;OATL3; PAX2; PCBD; PEO1; PHYH; PNLIP; PSAP; PTEN; RBP4; RDPA; RET;SFTPA1; SFTPD; SHFM3; SIAL; THC2; TLX1; TNFRSF6; UFS; UROS; AA; ABCC8;ACAT1; ALX4; AMPD3; ANC; APOA1; APOA4; APOC3; ATM; BSCL2; BWS; CALCA;CAT; CCND1; CD3E; CD3G; CD59; CDKN1C; CLN2; CNTF; CPT1A; CTSC; DDB1;DDB2; DHCR7; DLAT; DRD4; ECB2; ED4; EVR1; EXT2; F2; FSHB; FTH1; G6PT1;G6PT2; GIF; HBB; HBBP1; HBD; HBE1; HBG1; HBG2; HMBS; HND; HOMG2; HRAS;HVBS1; IDDM2; IGER; INS; JBS; KCNJ11; KCNJ1; KCNQ1; LDHA; LRP5; MEN1;MLL; MYBPC3; MYO7A; NNO1; OPPG; OPTB1; PAX6; PC; PDX1; PGL2; PGR; PORC;PTH; PTS; PVRL1; PYGM; RAG1; RAG2; ROM1; RRAS2; SAA1; SCA5; SCZD2; SDHD;SERPING1; SMPD1; TCIRG1; TCL2; TECTA; TH; TREH; TSG101; TYR; USH1C;VMD2; VRNI; WT1; WT2; ZNF145; A2M; AAAS; ACADS; ACLS; ACVRL1; ALDH2;AMHR2; AOM; AQP2; ATD; ATP2A2; BDC; C1R; CD4; CDK4; CNA1; COL2A1;CYP27B1; DRPLA; ENUR2; FEOM1; FGF23; FPF; GNB3; GNS; HAL; HBP1; HMGA2;HMN2; HPD; IGF1; KCNA1; KERA; KRAS2; KRT1; KRT2A; KRT3; KRT4; KRT5;KRT6A; KRT6B; KRTHB6; LDHB; LYZ; MGCT; MPE; MVK; MYL2; OAP; PAH; PPKB;PRB3; PTPN11; PXR1; RLS; RSN; SAS; SAX1; SCA2; SCNN1A; SMAL; SPPM;SPSMA; TBX3; TBX5; TCF1; TPI1; TSC3; ULR; VDR; VWF; ATP7B; BRCA2; BRCD1;CLN5; CPB2; ED2; EDNRB; ENUR1; ERCC5; F10; F7; GJB2; GJB6; IPF1; MBS1;MCOR; NYS4; PCCA; RB1; RHOK; SCZD7; SGCG; SLC10A2; SLC25A15; STARP1;ZNF198; ACHM1; ARVD1; BCH; CTAA1; DAD1; DFNB5; EML1; GALC; GCH1; IBGC1;IGH@; IGHC group; IGHG1; IGHM; IGHR; IV; LTBP2; MCOP; MJD; MNG1; MPD1;MPS3C; MYH6; MYH7; NP; NPC2; PABPN1; PSEN1; PYGL; RPGRIP1; SERPINA1;SERPINA3; SERPINA6; SLC7A7; SPG3A; SPTB; TCL1A; TGM1; TITF1; TMIP; TRA@;TSHR; USH1A; VP; ACCPN; AHO2; ANCR; B2M; BBS4; BLM; CAPN3; CDAN1; CDAN3;CLN6; CMH3; CYP19; CYP1A1; CYP1A2; DYX1; EPB42; ETFA; EYCL3; FAH; FBN1;FES; HCVS; HEXA; IVD; LCS1; LIPC; MY05A; OCA2; OTSC1; PWCR; RLBP1;SLC12A1; SPG6; TPM1; UBE3A; WMS; ABCC6; ALDOA; APRT; ATP2A1; BBS2;CARD15; CATM; CDH1; CETP; CHST6; CLN3; CREBBP; CTH; CTM; CYBA; CYLD;DHS; DNASE1; DPEP1; ERCC4; FANCA; GALNS; GAN; HAGH; HBA1; HBA2; HBHR;HBQ1; HBZ; HBZP; HP; HSD11B2; IL4R; LIPB; MC1R; MEFV; MHC2TA; MLYCD;MMVP1; PHKB; PHKG2; PKD1; PKDTS; PMM2; PXE; SALL1; SCA4; SCNN1B; SCNN1G;SLC12A3; TAT; TSC2; VDI; WT3; ABR; ACACA; ACADVL; ACE; ALDH3A2; APOH;ASPA; AXIN2; BCL5; BHD; BLMH; BRCA1; CACD; CCA1; CCZS; CHRNB1; CHRNE;CMT1A; COL1A1; CORDS; CTNS; EPX; ERBB2; G6PC; GAA; GALK1; GCGR; GFAP;GH1; GH2; GP1BA; GPSC; GUCY2D; ITGA2B; ITGB3; ITGB4; KRT10; KRT12;KRT13; KRT14; KRT14L1; KRT14L2; KRT14L3; KRT16; KRT16L1; KRT16L2; KRT17;KRT9; MAPT; MDB; MDCR; MGI; MHS2; MKS1; MPO; MYO15A; NAGLU; NAPB; NF1;NME1; P4HB; PAFAH1B1; PECAM1; PEX12; PHB; PMP22; PRKAR1A; PRKCA;PRKWNK4; PRP8; PRPF8; PTLAH; RARA; RCV1; RMSA1; RP17; RSS; SCN4A;SERPINF2; SGCA; SGSH; SHBG; SLC2A4; SLC4A1; SLC6A4; SMCR; SOST; SOX9;SSTR2; SYM1; SYNS1; TCF2; THRA; TIMP2; TOC; TOP2A; TP53; TRIM37; VBCH;ATP8B1; BCL2; CNSN; CORD1; CYB5; DCC; F5F8D; FECH; FEO; LAMA3; LCFS2;MADH4; MAFD1; MC2R; MCL; MYP2; NPC1; SPPK; TGFBRE; TGIF; TTR; AD2; AMH;APOC2; APOE; ATHS; BAX; BCKDHA; BCL3; BFIC; C3; CACNA1A; CCO; CEACAM5;COMP; CRX; DBA; DDU; DFNA4; DLL3; DM1; DMWD; E11S; ELA2; EPOR; ERCC2;ETFB; EXT3; EYCL1; FTL; FUT1; FUT2; FUT6; GAMT; GCDH; GPI; GUSM; HB1;HCL1; HHC2; HHC3; ICAM3; INSR; JAK3; KLK3; LDLR; LHB; LIG1; LOH19CR1;LYL1; MAN2B1; MCOLN1; MDRV; MLLT1; NOTCH3; NPHS1; OFC3; OPA3; PEPD;PRPF31; PRTN3; PRX; PSG1; PVR; RYR1; SLC5A5; SLC7A9; STK11; TBXA2R;TGFB1; TNNI3; TYROBP; ADA; AHCY; AVP; CDAN2; CDPD1; CHED1; CHED2;CHRNA4; CST3; EDN3; EEGV1; FTLL1; GDF5; GNAS; GSS; HNF4A; JAG1; KCNQ2;MKKS; NBIA1; PCK1; PI3; PPCD; PPGB; PRNP; THBD; TOP1; AIRE; APP; CBS;COL6A1; COL6A2; CSTB; DCR; DSCR1; FPDMM; HLCS; HPE1; ITGB2; KCNE1; KNO;PRSS7; RUNX1; SOD1; TAM; ADSL; ARSA; BCR; CECR; CHEK2; COMT; CRYBB2;CSF2RB; CTHM; CYP2D6; CYP2D7P1; DGCR; DIA1; EWSR1; GGT1; MGCR; MN1;NAGA; NF2; OGS2; PDGFB; PPARA; PRODH; SCO2; SCZD4; SERPIND1; SLC5A1;SOX10; TCN2; TIMP3; TST; VCF; ABCD1; ACTL1; ADFN; AGMX2; AHDS; AIC;AIED; AIH3; ALAS2; AMCD; AMELX; ANOP1; AR; ARAF1; ARSC2; ARSE; ARTS;ARX; ASAT; ASSP5; ATP7A; ATRX; AVPR2; BFLS; BGN; BTK; BZX; C1HR;CACNA1F; CALB3; CBBM; CCT; CDR1; CFNS; CGF1; CHM; CHR39c; CIDX; CLA2;CLCN5; CLS; CMTX2; CMTX3; CND; COD1; COD2; COL4A5; COL4A6; CPX; CVD1;CYBB; DCX; DFN2; DFN4; DFN6; DHOF; DIAPH2; DKC1; DMD; DSS; DYT3; EBM;EBP; ED1; ELK1; EMD; EVR2; F8; F9; FCP1; FDPSL5; FGD1; FGS1; FMR1; FMR2;G6PD; GABRA3; GATA1; GDI1; GDXY; GJB1; GK; GLA; GPC3; GRPR; GTD; GUST;HMS1; HPRT1; HPT; HTC2; HTR2c; HYR; IDS; IHG1; IL2RG; INDX; IP1; IP2;JMS; KAL1; KFSD; L1CAM; LAMP2; MAA; MAFD2; MAOA; MAOB; MCF2; MCS; MEAX;MECP2; MF4; MGC1; MIC5; MID1; MLLT7; MLS; MRSD; MRX14; MRX1; MRX20;MRX2; MRX3; MRX40; MRXA; MSD; MTM1; MYCL2; MYP1; NDP; NHS; NPHL1; NROB1;NSX; NYS1; NYX; OA1; OASD; OCRL; ODT1; OFD1; OPA2; OPD1; OPEM; OPN1LW;OPN1MW; OTC; P3; PDHA1; PDR; PFC; PFKFB1; PGK1; PGK1P1; PGS; PHEX;PHKA1; PHKA2; PHP; PIGA; PLP1; POF1; POLA; POU3F4; PPMX; PRD; PRPS1;PRPS2; PRS; RCCP2; RENBP; RENS1; RP2; RP6; RPGR; RPS4X; RPS6KA3; RS1;511; SDYS; SEDL; SERPINA7; SH2D1A; SHFM2; SLC25A5; SMAX2; SRPX; SRS;STS; SYN1; SYP; TAF1; TAZ; TBX22; TDD; TFE3; THAS; THC; TIMM8A; TIMP1;TKCR; TNFSF5; UBE1; UBE2A; WAS; WSN; WTS; WWS; XIC; XIST; XK; XM; XS;ZFX; ZIC3; ZNF261; ZNF41; ZNF6; AMELY; ASSP6; AZF1; AZF2; DAZ; GCY;RPS4Y; SMCY; SRY; ZFY; ABAT; AEZ; AFA; AFD1; ASAH1; ASD1; ASMT; CCAT;CECR9; CEPA; CLA3; CLN4; CSF2RA; CTS1; DF; DIH1; DWS; DYT2; DYT4; EBR3;ECT; EEF1A1L14; EYCL2; FANCB; GCSH; GCSL; GIP; GTS; HHG; HMI; HOAC;HOKPP2; HRPT1; HSD3B3; HTC1; HV1S; ICHQ; ICR1; ICR5; IL3RA; KAL2; KMS;KRT18; KSS; LCAT; LHON; LIMM; MANBB; MCPH2; MEB; MELAS; MIC2; MPFD; MS;MSS; MTATP6; MTCO1; MTCO3; MTCYB; MTND1; MTND2; MTND4; MTND5; MTND6;MTRNR1; MTRNR2; MTTE; MTTG; MTTI; MTTK; MTTL1; MTTL2; MTTN; MTTP; MTTS1;NAMSD; OCD1; OPD2; PCK2; PCLD; PCOS1; PFKM; PKD3; PRCA1; PRO1; PROP1;RBS; RFXAP; RP; SHOX; SLC25A6; SPG5B; STO; SUOX; THM; or TTD. Eachrecombinant protein represents a separate embodiment of the presentinvention.

In another embodiment, the present invention provides a method fortreating anemia in a subject, comprising contacting a cell of thesubject with an in vitro-synthesized RNA molecule, the invitro-synthesized RNA molecule encoding erythropoietin, thereby treatinganemia in a subject. In another embodiment, the in vitro-synthesized RNAmolecule further comprises a pseudouridine or a modified nucleoside.Each possibility represents a separate embodiment of the presentinvention. In another embodiment, the cell is a subcutaneous tissuecell. In another embodiment, the cell is a lung cell. In anotherembodiment, the cell is a fibroblast. In another embodiment, the cell isa lymphocyte. In another embodiment, the cell is a smooth muscle cell.In another embodiment, the cell is any other type of cell known in theart. Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the present invention provides a method fortreating a vasospasm in a subject, comprising contacting a cell of thesubject with an in vitro-synthesized RNA molecule, the invitro-synthesized RNA molecule encoding inducible nitric oxide synthase(iNOS), thereby treating a vasospasm in a subject.

In another embodiment, the present invention provides a method forimproving a survival rate of a cell in a subject, comprising contactingthe cell with an in vitro-synthesized RNA molecule, the invitro-synthesized RNA molecule encoding a heat shock protein, therebyimproving a survival rate of a cell in a subject.

In another embodiment, the cell whose survival rate is improved is anischemic cell. In another embodiment, the cell is not ischemic. Inanother embodiment, the cell has been exposed to an ischemicenvironment. In another embodiment, the cell has been exposed to anenvironmental stress. Each possibility represents a separate embodimentof the present invention.

In another embodiment, the present invention provides a method fordecreasing an incidence of a restenosis of a blood vessel following aprocedure that enlarges the blood vessel, comprising contacting a cellof the blood vessel with an in vitro-synthesized RNA molecule, the invitro-synthesized RNA molecule encoding a heat shock protein, therebydecreasing an incidence of a restenosis in a subject.

In another embodiment, the procedure is an angioplasty. In anotherembodiment, the procedure is any other procedure known in the art thatenlarges the blood vessel. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides a method forincreasing a hair growth from a hair follicle is a scalp of a subject,comprising contacting a cell of the scalp with an in vitro-synthesizedRNA molecule, the in vitro-synthesized RNA molecule encoding atelomerase or an immunosuppressive protein, thereby increasing a hairgrowth from a hair follicle.

In another embodiment, the immunosuppressive protein isα-melanocyte-stimulating hormone (α-MSH). In another embodiment, theimmunosuppressive protein is transforming growth factor-β1 (TGF-β1). Inanother embodiment, the immunosuppressive protein is insulin-like growthfactor-I (IGF-I). In another embodiment, the immunosuppressive proteinis any other immunosuppressive protein known in the art. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a method ofinducing expression of an enzyme with antioxidant activity in a cell,comprising contacting the cell with an in vitro-synthesized RNAmolecule, the in vitro-synthesized RNA molecule encoding the enzyme,thereby inducing expression of an enzyme with antioxidant activity in acell.

In another embodiment, the enzyme is catalase. In another embodiment,the enzyme is glutathione peroxidase. In another embodiment, the enzymeis phospholipid hydroperoxide glutathione peroxidase. In anotherembodiment, the enzyme is superoxide dismutase-1. In another embodiment,the enzyme is superoxide dismutase-2. In another embodiment, the enzymeis any other enzyme with antioxidant activity that is known in the art.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the present invention provides a method fortreating cystic fibrosis in a subject, comprising contacting a cell ofthe subject with an in vitro-synthesized RNA molecule, the invitro-synthesized RNA molecule encoding Cystic Fibrosis TransmembraneConductance Regulator (CFTR), thereby treating cystic fibrosis in asubject.

In another embodiment, the present invention provides a method fortreating an X-linked agammaglobulinemia in a subject, comprisingcontacting a cell of the subject with an in vitro-synthesized RNAmolecule, the in vitro-synthesized RNA molecule encoding a Bruton'styrosine kinase, thereby treating an X-linked agammaglobulinemia.

In another embodiment, the present invention provides a method fortreating an adenosine deaminase severe combined immunodeficiency (ADASCID) in a subject, comprising contacting a cell of the subject with anin vitro-synthesized RNA molecule, the in vitro-synthesized RNA moleculeencoding an ADA, thereby treating an ADA SCID.

In another embodiment, the present invention provides a method forreducing immune responsiveness of the skin and improve skin pathology,comprising contacting a cell of the subject with an in vitro-synthesizedRNA molecule, the in vitro-synthesized RNA molecule encoding anecto-nucleoside triphosphate diphosphohydrolase, thereby reducing immuneresponsiveness of the skin and improve skin pathology.

In another embodiment, an RNA molecule or ribonucleotide molecule of thepresent invention is encapsulated in a nanoparticle. Methods fornanoparticle packaging are well known in the art, and are described, forexample, in Bose S, et al (Role of Nucleolin in Human ParainfluenzaVirus Type 3 Infection of Human Lung Epithelial Cells. J. Virol.78:8146. 2004); Dong Y et al.Poly(d,l-lactide-co-glycolide)/montmorillonite nanoparticles for oraldelivery of anticancer drugs. Biomaterials 26:6068. 2005); Lobenberg R.et al (Improved body distribution of 14C-labelled AZT bound tonanoparticles in rats determined by radioluminography. J Drug Target5:171. 1998); Sakuma S R et al (Mucoadhesion of polystyrenenanoparticles having surface hydrophilic polymeric chains in thegastrointestinal tract. Int J Pharm 177:161. 1999); Virovic L et al.Novel delivery methods for treatment of viral hepatitis: an update.Expert Opin Drug Deliv 2:707. 2005); and Zimmermann E et al,Electrolyte- and pH-stabilities of aqueous solid lipid nanoparticle(SLN) dispersions in artificial gastrointestinal media. Eur J PharmBiopharm 52:203. 2001). Each method represents a separate embodiment ofthe present invention.

Various embodiments of dosage ranges of compounds of the presentinvention can be used in methods of the present invention. In oneembodiment, the dosage is in the range of 1-10 μg/day. In anotherembodiment, the dosage is 2-10 μg/day. In another embodiment, the dosageis 3-10 μg/day. In another embodiment, the dosage is 5-10 μg/day. Inanother embodiment, the dosage is 2-20 μg/day. In another embodiment,the dosage is 3-20 μg/day. In another embodiment, the dosage is 5-20μg/day. In another embodiment, the dosage is 10-20 μg/day. In anotherembodiment, the dosage is 3-40 μg/day. In another embodiment, the dosageis 5-40 μg/day. In another embodiment, the dosage is 10-40 μg/day. Inanother embodiment, the dosage is 20-40 μg/day. In another embodiment,the dosage is 5-50 μg/day. In another embodiment, the dosage is 10-50μg/day. In another embodiment, the dosage is 20-50 μg/day. In oneembodiment, the dosage is 1-100 μg/day. In another embodiment, thedosage is 2-100 μg/day. In another embodiment, the dosage is 3-100μg/day. In another embodiment, the dosage is 5-100 μg/day. In anotherembodiment the dosage is 10-100 μg/day. In another embodiment the dosageis 20-100 μg/day. In another embodiment the dosage is 40-100 μg/day. Inanother embodiment the dosage is 60-100 μg/day.

In another embodiment, the dosage is 0.1 μg/day. In another embodiment,the dosage is 0.2 μg/day. In another embodiment, the dosage is 0.3μg/day. In another embodiment, the dosage is 0.5 μg/day. In anotherembodiment, the dosage is 1 μg/day. In another embodiment, the dosage is2 mg/day. In another embodiment, the dosage is 3 μg/day. In anotherembodiment, the dosage is 5 μg/day. In another embodiment, the dosage is10 μg/day. In another embodiment, the dosage is 15 μg/day. In anotherembodiment, the dosage is 20 μg/day. In another embodiment, the dosageis 30 μg/day.

In another embodiment, the dosage is 40 μg/day. In another embodiment,the dosage is 60 μg/day. In another embodiment, the dosage is 80 μg/day.In another embodiment, the dosage is 100 μg/day.

In another embodiment, the dosage is 10 μg/dose. In another embodiment,the dosage is 20 μg/dose. In another embodiment, the dosage is 30μg/dose. In another embodiment, the dosage is 40 μg/dose. In anotherembodiment, the dosage is 60 μg/dose. In another embodiment, the dosageis 80 μg/dose. In another embodiment, the dosage is 100 μg/dose. Inanother embodiment, the dosage is 150 μg/dose. In another embodiment,the dosage is 200 μg/dose. In another embodiment, the dosage is 300μg/dose. In another embodiment, the dosage is 400 μg/dose. In anotherembodiment, the dosage is 600 μg/dose. In another embodiment, the dosageis 800 μg/dose. In another embodiment, the dosage is 1000 μg/dose. Inanother embodiment, the dosage is 1.5 mg/dose. In another embodiment,the dosage is 2 mg/dose. In another embodiment, the dosage is 3 mg/dose.In another embodiment, the dosage is 5 mg/dose. In another embodiment,the dosage is 10 mg/dose. In another embodiment, the dosage is 15mg/dose. In another embodiment, the dosage is 20 mg/dose. In anotherembodiment, the dosage is 30 mg/dose. In another embodiment, the dosageis 50 mg/dose. In another embodiment, the dosage is 80 mg/dose. Inanother embodiment, the dosage is 100 mg/dose.

In another embodiment, the dosage is 10-20 μg/dose. In anotherembodiment, the dosage is 20-30 μg/dose. In another embodiment, thedosage is 20-40 μg/dose. In another embodiment, the dosage is 30-60μg/dose. In another embodiment, the dosage is 40-80 μg/dose. In anotherembodiment, the dosage is 50-100 μg/dose. In another embodiment, thedosage is 50-150 μg/dose. In another embodiment, the dosage is 100-200μg/dose. In another embodiment, the dosage is 200-300 μg/dose. Inanother embodiment, the dosage is 300-400 μg/dose. In anotherembodiment, the dosage is 400-600 μg/dose. In another embodiment, thedosage is 500-800 μg/dose. In another embodiment, the dosage is 800-1000μg/dose. In another embodiment, the dosage is 1000-1500 μg/dose. Inanother embodiment, the dosage is 1500-2000 μg/dose. In anotherembodiment, the dosage is 2-3 mg/dose. In another embodiment, the dosageis 2-5 mg/dose. In another embodiment, the dosage is 2-10 mg/dose. Inanother embodiment, the dosage is 2-20 mg/dose. In another embodiment,the dosage is 2-30 mg/dose. In another embodiment, the dosage is 2-50mg/dose. In another embodiment, the dosage is 2-80 mg/dose. In anotherembodiment, the dosage is 2-100 mg/dose. In another embodiment, thedosage is 3-10 mg/dose. In another embodiment, the dosage is 3-20mg/dose. In another embodiment, the dosage is 3-30 mg/dose. In anotherembodiment, the dosage is 3-50 mg/dose. In another embodiment, thedosage is 3-80 mg/dose. In another embodiment, the dosage is 3-100mg/dose. In another embodiment, the dosage is 5-10 mg/dose. In anotherembodiment, the dosage is 5-20 mg/dose. In another embodiment, thedosage is 5-30 mg/dose. In another embodiment, the dosage is 5-50mg/dose. In another embodiment, the dosage is 5-80 mg/dose. In anotherembodiment, the dosage is 5-100 mg/dose. In another embodiment, thedosage is 10-20 mg/dose. In another embodiment, the dosage is 10-30mg/dose. In another embodiment, the dosage is 10-50 mg/dose. In anotherembodiment, the dosage is 10-80 mg/dose. In another embodiment, thedosage is 10-100 mg/dose.

In another embodiment, the dosage is a daily dose. In anotherembodiment, the dosage is a weekly dose. In another embodiment, thedosage is a monthly dose. In another embodiment, the dosage is an annualdose. In another embodiment, the dose is one is a series of a definednumber of doses. In another embodiment, the dose is a one-time dose. Asdescribed below, in another embodiment, an advantage of RNA,oligoribonucleotide, or polyribonucleotide molecules of the presentinvention is their greater potency, enabling the use of smaller doses.

In another embodiment, the present invention provides a method forproducing a recombinant protein, comprising contacting an in vitrotranslation apparatus with an in vitro-synthesized oligoribonucleotide,the in vitro-synthesized oligoribonucleotide comprising a pseudouridineor a modified nucleoside, thereby producing a recombinant protein.

In another embodiment, the present invention provides a method forproducing a recombinant protein, comprising contacting an in vitrotranslation apparatus with an in vitro-transcribed RNA molecule of thepresent invention, the in vitro-transcribed RNA molecule comprising apseudouridine or a modified nucleoside, thereby producing a recombinantprotein.

In another embodiment, the present invention provides an in vitrotranscription apparatus, comprising: an unmodified nucleotide, anucleotide containing a pseudouridine or a modified nucleoside, and apolymerase. In another embodiment, the present invention provides an invitro transcription kit, comprising: an unmodified nucleotide, anucleotide containing a pseudouridine or a modified nucleoside, and apolymerase. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the in vitro translation apparatus comprises areticulocyte lysate. In another embodiment, the reticulocyte lysate is arabbit reticulocyte lysate.

In another embodiment, the present invention provides a method ofreducing an immunogenicity of an oligoribonucleotide molecule or RNAmolecule, the method comprising the step of replacing a nucleotide ofthe oligoribonucleotide molecule or RNA molecule with a modifiednucleotide that contains a modified nucleoside or a pseudouridine,thereby reducing an immunogenicity of an oligoribonucleotide molecule orRNA molecule.

In another embodiment, the present invention provides a method ofreducing an immunogenicity of a gene-therapy vector comprising apolyribonucleotide molecule or RNA molecule, the method comprising thestep of replacing a nucleotide of the polyribonucleotide molecule or RNAmolecule with a modified nucleotide that contains a modified nucleosideor a pseudouridine, thereby reducing an immunogenicity of a gene-therapyvector.

In another embodiment, the present invention provides a method ofenhancing in vitro translation from an oligoribonucleotide molecule orRNA molecule, the method comprising the step of replacing a nucleotideof the oligoribonucleotide molecule or RNA molecule with a modifiednucleotide that contains a modified nucleoside or a pseudouridine,thereby enhancing in vitro translation from an oligoribonucleotidemolecule or RNA molecule.

In another embodiment, the present invention provides a method ofenhancing in vivo translation from a gene-therapy vector comprising apolyribonucleotide molecule or RNA molecule, the method comprising thestep of replacing a nucleotide of the polyribonucleotide molecule or RNAmolecule with a modified nucleotide that contains a modified nucleosideor a pseudouridine, thereby enhancing in vivo translation from agene-therapy vector.

In another embodiment, the present invention provides a method ofincreasing efficiency of delivery of a recombinant protein by a genetherapy vector comprising a polyribonucleotide molecule or RNA molecule,the method comprising the step of replacing a nucleotide of thepolyribonucleotide molecule or RNA molecule with a modified nucleotidethat contains a modified nucleoside or a pseudouridine, therebyincreasing efficiency of delivery of a recombinant protein by a genetherapy vector.

In another embodiment, the present invention provides a method ofincreasing in vivo stability of gene therapy vector comprising apolyribonucleotide molecule or RNA molecule, the method comprising thestep of replacing a nucleotide of the polyribonucleotide molecule or RNAmolecule with a modified nucleotide that contains a modified nucleosideor a pseudouridine, thereby increasing in vivo stability of gene therapyvector.

In another embodiment, the present invention provides a method ofsynthesizing an in vitro-transcribed RNA molecule comprising apseudouridine nucleoside, comprising contacting an isolated polymerasewith a mixture of unmodified nucleotides and the modified nucleotide(Examples 2 and 7).

In another embodiment, in vitro transcription methods of the presentinvention utilize an extract from an animal cell. In another embodiment,the extract is from a reticulocyte or cell with similar efficiency of invitro transcription. In another embodiment, the extract is from anyother type of cell known in the art. Each possibility represents aseparate embodiment of the present invention.

Any of the RNA molecules or oligoribonucleotide molecules of the presentinvention may be used, in another embodiment, in any of the methods ofthe present invention.

In another embodiment, the present invention provides a method ofenhancing an immune response to an antigen, comprising administering theantigen in combination with mitochondrial (mt) RNA (Examples 1 and 5).

In another embodiment, the present invention provides a method ofreducing the ability of an RNA molecule to stimulate a dendritic cell(DC), comprising modifying a nucleoside of the RNA molecule by a methodof the present invention (Examples).

In another embodiment, the DC is a DC1 cell. In another embodiment, theDC is a DC2 cell. In another embodiment, the DC is a subtype of a DC1cell or DC2 cell. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, the present invention provides a method ofreducing the ability of an RNA molecule to stimulate signaling by TLR3,comprising modifying a nucleoside of the RNA molecule by a method of thepresent invention. In another embodiment, the present invention providesa method of reducing the ability of an RNA molecule to stimulatesignaling by TLR7, comprising modifying a nucleoside of the RNA moleculeby a method of the present invention. In another embodiment, the presentinvention provides a method of reducing the ability of an RNA moleculeto stimulate signaling by TLR8, comprising modifying a nucleoside of theRNA molecule by a method of the present invention. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, all the inter-nucleotide linkages in the RNA,oligoribonucleotide, or polyribonucleotide molecule are phosphodiester.In another embodiment, the inter-nucleotide linkages are predominantlyphosphodiester. In another embodiment, most of the inter-nucleotidelinkages are phosphorothioate. In another embodiment, most theinter-nucleotide linkages are phosphodiester. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the percentage of the inter-nucleotide linkagesthat are phosphodiester is above 50%. In another embodiment, thepercentage is above 10%. In another embodiment, the percentage is above15%. In another embodiment, the percentage is above 20%. In anotherembodiment, the percentage is above 25%. In another embodiment, thepercentage is above 30%. In another embodiment, the percentage is above35%. In another embodiment, the percentage is above 40%. In anotherembodiment, the percentage is above 45%. In another embodiment, thepercentage is above 55%. In another embodiment, the percentage is above60%. In another embodiment, the percentage is above 65%. In anotherembodiment, the percentage is above 70%. In another embodiment, thepercentage is above 75%. In another embodiment, the percentage is above80%. In another embodiment, the percentage is above 85%. In anotherembodiment, the percentage is above 90%. In another embodiment, thepercentage is above 95%.

In another embodiment, a method of the present invention comprisesincreasing the number, percentage, or frequency of modified nucleosidesin the RNA molecule to decrease immunogenicity or increase efficiency oftranslation. As provided herein, the number of modified residues in anRNA, oligoribonucleotide, or polyribonucleotide molecule determines, inanother embodiment, the magnitude of the effects observed in the presentinvention.

In another embodiment, the present invention provides a method forintroducing a recombinant protein into a cell of a subject, comprisingcontacting the subject with an in vitro-transcribed RNA moleculeencoding the recombinant protein, the in vitro-transcribed RNA moleculefurther comprising a modified nucleoside, thereby introducing arecombinant protein into a cell of a subject.

In another embodiment, the present invention provides a method fordecreasing TNF-α production in response to a gene therapy vector in asubject, comprising the step of engineering the vector to contain apseudouridine or a modified nucleoside base, thereby decreasing TNF-αproduction in response to a gene therapy vector in a subject.

In another embodiment, the present invention provides a method fordecreasing IL-12 production in response to a gene therapy vector in asubject, comprising the step of engineering the vector to contain apseudouridine or a modified nucleoside base, thereby decreasing IL-12production in response to a gene therapy vector in a subject.

In another embodiment, the present invention provides a method ofreducing an immunogenicity of a gene therapy vector, comprisingintroducing a modified nucleoside into said gene therapy vector, therebyreducing an immunogenicity of a gene therapy vector.

As provided herein, findings of the present invention show that primaryDC have an additional RNA signaling entity that recognizes m5C- andm6A-modified RNA and whose signaling is inhibited by modification of Uresidues.

In another embodiment, an advantage of an RNA, oligoribonucleotide, andpolyribonucleotide molecules of the present invention is that RNA doesnot incorporate to the genome (as opposed to DNA-based vectors). Inanother embodiment, an advantage is that translation of RNA, andtherefore appearance of the encoded product, is instant. In anotherembodiment, an advantage is that the amount of protein generated fromthe mRNA can be regulated by delivering more or less RNA. In anotherembodiment, an advantage is that repeated delivery of unmodified RNAcould induce autoimmune reactions.

In another embodiment, an advantage is lack of immunogenicity, enablingrepeated delivery without generation of inflammatory cytokines.

In another embodiment, stability of RNA is increased by circularization,decreasing degradation by exonucleases.

In another embodiment, the present invention provides a method oftreating a subject with a disease that comprises an immune responseagainst a self-RNA molecule, comprising administering to the subject anantagonist of a TLR-3 molecule, thereby treating a subject with adisease that comprises an immune response against a self-RNA molecule.

In another embodiment, the present invention provides a method oftreating a subject with a disease that comprises an immune responseagainst a self-RNA molecule, comprising administering to the subject anantagonist of a TLR-7 molecule, thereby treating a subject with adisease that comprises an immune response against a self-RNA molecule.

In another embodiment, the present invention provides a method oftreating a subject with a disease that comprises an immune responseagainst a self-RNA molecule, comprising administering to the subject anantagonist of a TLR-8 molecule, thereby treating a subject with adisease that comprises an immune response against a self-RNA molecule.

In another embodiment, the disease that comprises an immune responseagainst a self-RNA molecule is an auto-immune disease. In anotherembodiment, the disease is systemic lupus erythematosus (SLE). Inanother embodiment, the disease is another disease known in the art thatcomprises an immune response against a self-RNA molecule. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the present invention provides a kit comprising areagent utilized in performing a method of the present invention. Inanother embodiment, the present invention provides a kit comprising acomposition, tool, or instrument of the present invention.

In another embodiment, the present invention provides a kit formeasuring or studying signaling by a TLR3, TLR7 and TLR8 receptor, asexemplified in Example 4.

In another embodiment, a treatment protocol of the present invention istherapeutic. In another embodiment, the protocol is prophylactic. Eachpossibility represents a separate embodiment of the present invention.

In one embodiment, the phrase “contacting a cell” or “contacting apopulation” refers to a method of exposure, which can be direct orindirect. In one method such contact comprises direct injection of thecell through any means well known in the art, such as microinjection. Inanother embodiment, supply to the cell is indirect, such as viaprovision in a culture medium that surrounds the cell, or administrationto a subject, or via any route known in the art. In another embodiment,the term “contacting” means that the molecule of the present inventionis introduced into a subject receiving treatment, and the molecule isallowed to come in contact with the cell in vivo. Each possibilityrepresents a separate embodiment of the present invention.

Methods for quantification of reticulocyte frequency and for measuringEPO biological activity are well known in the art, and are described,for Example, in Ramos, A S et al (Biological evaluation of recombinanthuman erythropoietin in pharmaceutical products. Braz J Med Biol Res36:1561). Each method represents a separate embodiment of the presentinvention.

Compositions of the present invention can be, in another embodiment,administered to a subject by any method known to a person skilled in theart, such as parenterally, paracancerally, transmucosally,transdermally, intramuscularly, intravenously, intra-dermally,subcutaneously, intra-peritonealy, intra-ventricularly, intra-cranially,intra-vaginally or intra-tumorally.

In another embodiment of methods and compositions of the presentinvention, the compositions are administered orally, and are thusformulated in a form suitable for oral administration, i.e. as a solidor a liquid preparation. Suitable solid oral formulations includetablets, capsules, pills, granules, pellets and the like. Suitableliquid oral formulations include solutions, suspensions, dispersions,emulsions, oils and the like. In another embodiment of the presentinvention, the active ingredient is formulated in a capsule. Inaccordance with this embodiment, the compositions of the presentinvention comprise, in addition to the active compound and the inertcarrier or diluent, a hard gelating capsule.

In other embodiments, the pharmaceutical compositions are administeredby intravenous, intra-arterial, or intra-muscular injection of a liquidpreparation. Suitable liquid formulations include solutions,suspensions, dispersions, emulsions, oils and the like. In anotherembodiment, the pharmaceutical compositions are administeredintravenously and are thus formulated in a form suitable for intravenousadministration. In another embodiment, the pharmaceutical compositionsare administered intra-arterially and are thus formulated in a formsuitable for intra-arterial administration. In another embodiment, thepharmaceutical compositions are administered intra-muscularly and arethus formulated in a form suitable for intra-muscular administration.

In another embodiment, the pharmaceutical compositions are administeredtopically to body surfaces and are thus formulated in a form suitablefor topical administration. Suitable topical formulations include gels,ointments, creams, lotions, drops and the like. For topicaladministration, the compositions or their physiologically toleratedderivatives are prepared and applied as solutions, suspensions, oremulsions in a physiologically acceptable diluent with or without apharmaceutical carrier.

In another embodiment, the composition is administered as a suppository,for example a rectal suppository or a urethral suppository. In anotherembodiment, the pharmaceutical composition is administered bysubcutaneous implantation of a pellet. In another embodiment, the pelletprovides for controlled release of agent over a period of time.

In another embodiment, the active compound is delivered in a vesicle,e.g. a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al.,in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989);Lopez-Berestein, ibid., pp. 317-327; see generally ibid).

As used herein “pharmaceutically acceptable carriers or diluents” arewell known to those skilled in the art. The carrier or diluent may bemay be, in various embodiments, a solid carrier or diluent for solidformulations, a liquid carrier or diluent for liquid formulations, ormixtures thereof.

In another embodiment, solid carriers/diluents include, but are notlimited to, a gum, a starch (e.g. corn starch, pregeletanized starch), asugar (e.g., lactose, mannitol, sucrose, dextrose), a cellulosicmaterial (e.g. microcrystalline cellulose), an acrylate (e.g.polymethylacrylate), calcium carbonate, magnesium oxide, talc, ormixtures thereof.

In other embodiments, pharmaceutically acceptable carriers for liquidformulations may be aqueous or non-aqueous solutions, suspensions,emulsions or oils. Examples of non-aqueous solvents are propyleneglycol, polyethylene glycol, and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions,emulsions or suspensions, including saline and buffered media. Examplesof oils are those of petroleum, animal, vegetable, or synthetic origin,for example, peanut oil, soybean oil, mineral oil, olive oil, sunfloweroil, and fish-liver oil.

Parenteral vehicles (for subcutaneous, intravenous, intraarterial, orintramuscular injection) include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's and fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers such as those based on Ringer's dextrose, andthe like. Examples are sterile liquids such as water and oils, with orwithout the addition of a surfactant and other pharmaceuticallyacceptable adjuvants. In general, water, saline, aqueous dextrose andrelated sugar solutions, and glycols such as propylene glycols orpolyethylene glycol are preferred liquid carriers, particularly forinjectable solutions. Examples of oils are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil,mineral oil, olive oil, sunflower oil, and fish-liver oil.

In another embodiment, the compositions further comprise binders (e.g.acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone),disintegrating agents (e.g. cornstarch, potato starch, alginic acid,silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodiumstarch glycolate), buffers (e.g., Tris-HCI., acetate, phosphate) ofvarious pH and ionic strength, additives such as albumin or gelatin toprevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80,Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g.sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g.,glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid,sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g.hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosityincreasing agents (e.g. carbomer, colloidal silicon dioxide, ethylcellulose, guar gum), sweeteners (e.g. aspartame, citric acid),preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants(e.g. stearic acid, magnesium stearate, polyethylene glycol, sodiumlauryl sulfate), flow-aids (e.g. colloidal silicon dioxide),plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers(e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymercoatings (e.g., poloxamers or poloxamines), coating and film formingagents (e.g. ethyl cellulose, acrylates, polymethacrylates) and/oradjuvants. Each of the above excipients represents a separate embodimentof the present invention.

In another embodiment, the pharmaceutical compositions provided hereinare controlled-release compositions, i.e. compositions in which thecompound is released over a period of time after administration.Controlled- or sustained-release compositions include formulation inlipophilic depots (e.g. fatty acids, waxes, oils). In anotherembodiment, the composition is an immediate-release composition, i.e. acomposition in which the entire compound is released immediately afteradministration.

In another embodiment, molecules of the present invention are modifiedby the covalent attachment of water-soluble polymers such aspolyethylene glycol, copolymers of polyethylene glycol and polypropyleneglycol, carboxymethyl cellulose, dextran, polyvinyl alcohol,polyvinylpyrrolidone or polyproline. The modified compounds are known toexhibit substantially longer half-lives in blood following intravenousinjection than do the corresponding unmodified compounds (Abuchowski etal., 1981; Newmark et al., 1982; and Katre et al., 1987). Suchmodifications also increase, in another embodiment, the compound'ssolubility in aqueous solution, eliminate aggregation, enhance thephysical and chemical stability of the compound, and greatly reduce theimmunogenicity and reactivity of the compound. As a result, the desiredin vivo biological activity may be achieved by the administration ofsuch polymer-compound abducts less frequently or in lower doses thanwith the unmodified compound.

An active component is, in another embodiment, formulated into thecomposition as neutralized pharmaceutically acceptable salt forms.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide or antibodymolecule), which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed from the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

Each of the above additives, excipients, formulations and methods ofadministration represents a separate embodiment of the presentinvention.

EXPERIMENTAL DETAILS SECTION Example 1 Naturally Occurring RNA MoleculesExhibit Differential Abilities to Activate Dendritic Cells Materials andExperimental Methods

Plasmids and Reagents

Plasmids pT7T3D-MART-1 and pUNO-hTLR3 were obtained from the ATCC(Manassas, Va.) and InvivoGen (San Diego, Calif.), respectively. pTEVlucwas obtained from Dr Daniel Gallie (UC Riverside), contains pT7-TEV (theleader sequence of the tobacco etch viral genomic RNA)-luciferase-A50,and is described in Gallie, D R et al, 1995. The tobacco etch viral 5′leader and poly(A) tail are functionally synergistic regulators oftranslation. Gene 165:233) pSVren was generated from p2luc (GrentzmannG, Ingram J A, et al, A dual-luciferase reporter system for studyingrecoding signals. RNA 1998; 4(4): 479-86) by removal of the fireflyluciferase coding sequence with BamHI and NotI digestions, end-filling,and religation.

Human TLR3-specific siRNA, pTLR3-sh was constructed by insertingsynthetic ODN encoding shRNA with 20-nt-long homology to human TLR3 (nt703-722, accession: NM_(—)003265) into plasmid pSilencer 4.1-CMV-neo(Ambion, Austin, Tex.). pCMV-hTLR3 was obtained by first cloninghTLR3-specific PCR product (nt 80-2887; Accession NM_(—)003265) intopCRII-TOPO (Invitrogen, Carlsbad, Calif.), then released with Nhe I-HindIII cutting and subcloning to the corresponding sites of pcDNA3.1(Invitrogen). LPS (E. coli 055:B5) was obtained from Sigma Chemical Co,St. Louis, Mo. CpG ODN-2006 and R-848 were obtained from InvivoGen.

Cells and Cell Culture

Human embryonic kidney 293 cells (ATCC) were propagated in DMEMsupplemented with glutamine (Invitrogen) and 10% FCS (Hyclone, Ogden,Utah) (complete medium). In all cases herein, “293 cells” refers tohuman embryonic kidney (HEK) 293 cells. 293-hTLR3 cell line wasgenerated by transforming 293 cells with pUNO-hTLR3. Cell lines293-hTLR7, 293-hTLR8 and 293-hTLR9 (InvivoGen) were grown in completemedium supplemented with blasticidin (10 μg/ml) (Invivogen). Cell lines293-ELAM-luc and TLR7-293 (M. Lamphier, Eisai Research Institute,Andover Mass.), and TLR3-293 cells were cultured as described (Kariko etal, 2004, mRNA is an endogenous ligand for Toll-like receptor 3. J BiolChem 279: 12542-12550). Cell lines 293, 293-hTLR7 and 293-hTLR8 werestably transfected with pTLR3-sh and selected with G-418 (400 μg/ml)(Invitrogen). Neo-resistant colonies were screened and only those thatdid not express TLR3, determined as lack of IL-8 secretion in responseto poly(I):(C), were used in further studies. Leukopheresis samples wereobtained from HIV-uninfected volunteers through an IRB-approvedprotocol.

Murine DC Generation

Murine DC were generated by collecting bone marrow cells from the tibiaand femurs of 6-8-week-old C57BL/6 mice and lysing the red blood cells.Cells were seeded in 6-well plates at 10⁶ cells/well in 2 ml DMEM+10%FCS and 20 ng/ml muGM-CSF (R & D Systems). On day 3, 2 ml of freshmedium with muGM-CSF was added. On day 6, 2 ml medium/well wascollected, and cells were pelleted and resuspended in fresh medium withmuGM-CSF. On day 7 of the culture, the muDC were harvested, washed.

Natural RNA

Mitochondria were isolated from platelets obtained from the Universityof Pennsylvania Blood Bank using a fractionation lyses procedure(Mitochondria isolation kit; Pierce, Rockford, Ill.). RNA was isolatedfrom the purified mitochondria, cytoplasmic and nuclear fractions of 293cells, un-fractioned 293 cells, rat liver, mouse cell line TUBO, andDHSalpha strain of E. coli by Master Blaster® (BioRad, Hercules,Calif.). Bovine tRNA, wheat tRNA, yeast tRNA, E. coli tRNA, poly(A)+mRNA from mouse heart and poly(I):(C) were purchased from Sigma, totalRNA from human spleen and E. coli RNA were purchased from Ambion.Oligoribonucleotide-5′-monophosphates were synthesized chemically(Dharmacon, Lafayette, Colo.).

Aliquots of RNA samples were incubated in the presence of Benzonasenuclease (1 Upper 5 μl of RNA at 1 microgram per microliter (μg/μl) for1 h) (Novagen, Madison, Wis.). Aliquots of RNA-730 were digested withalkaline phosphatase (New England Biolabs). RNA samples were analyzed bydenaturing agarose or polyacrylamide gel electrophoresis for qualityassurance. Assays for LPS in RNA preparations using the LimulusAmebocyte Lysate gel clot assay were negative with a sensitivity of 3picograms per milliliter (pg/ml) (University of Pennsylvania, CoreFacility).

HPLC Analysis

Nucleoside monophosphates were separated and visualized via HPLC. Torelease free nucleoside 3′-monophosphates, 5 μg aliquots of RNA weredigested with 0.1 U RNase T2 (Invitrogen) in 10 μl of 50 mM NaOAc and 2mM EDTA buffer (pH 4.5) overnight, then the samples were injected intoan Agilent 1100 HPLC using a Waters Symmetry C18 column (Waters,Milford, Mass.). At a flow rate of 1 mL/min, a gradient from 100% bufferA (30 mM KH₂PO₄ and 10 mM tetraethylammonium phosphate [PicA reagent,Waters], pH 6.0) to 30% buffer B (acetonitrile) was run over 60 minutes.Nucleotides were detected using a photodiode array at 254 nm. Identitieswere verified by retention times and spectra.

Dendritic Cell Assays

Dendritic cells in 96-well plates (approximately 1.1×10⁵ cells/well)were treated with R-848, Lipofectin®, or Lipofectin®-RNA for 1 h, thenthe medium was changed. At the end of 8 h (unless otherwise indicated),cells were harvested for either RNA isolation or flow cytometry, whilethe collected culture medium was subjected to cytokine ELISA. The levelsof IL-12 (p70) (BD Biosciences Pharmingen, San Diego, Calif.), IFN-α,TNF-α, and IL-8 (Biosource International, Camarillo, Calif.) weremeasured in supernatants by sandwich ELISA. Cultures were performed intriplicate or quadruplicate and measured in duplicate.

Northern Blot Analysis

RNA was isolated from MDDCs after an 8 h incubation following treatmentas described above. Where noted, cells were treated with 2.5 μg/mlcycloheximide (Sigma) 30 min prior to the stimulation and throughout theentire length of incubation. RNA samples were processed and analyzed onNorthern blots as described (Kariko et al, 2004, ibid) using human TNF-αand GAPDH probes derived from plasmids (pE4 and pHcGAP, respectively)obtained from ATCC.

Results

To determine the immuno-stimulatory potential of different cellular RNAsubtypes, RNA was isolated from different subcellular compartments—i.e.cytoplasm, nucleus and mitochondria. These RNA fractions, as well astotal RNA, tRNA and polyA-tail-selected mRNA, all from mammaliansources, were complexed to Lipofectin® and added to MDDC. Whilemammalian total, nuclear and cytoplasmic RNA all stimulated MDDC, asevidenced by detectable TNF-α secretion, the TNF-α levels were muchlower than those induced by in vitro-synthesized mRNA (FIG. 1).Moreover, mammalian tRNA did not induce any detectable level of TNF-α,while mitochondrial (mt) RNA induced much more TNF-α than the othermammalian RNA subtypes. Bacterial total RNA was also a potent activatorof MDDC; by contrast, bacterial tRNA induced only a low level of TNF-α.tRNA from other sources (yeast, wheat germ, bovine) werenon-stimulatory. Similar results were observed when RNA from othermammalian sources was tested. When RNA samples were digested withBenzonase, which cleaves ssRNA and dsRNA, RNA signaling was abolished inMDDC, verifying that TNF-α secretion was due to the RNA in thepreparations. The activation potentials of the RNA types testedexhibited an inverse correlation with the extent of nucleosidemodification. Similar results were obtained in the experiments describedin this Example for both types of cytokine-generated DC.

These findings demonstrate that the immunogenicity of RNA is affected bythe extent of nucleoside modification, with a greater degree ofmodification tending to decrease immunogenicity.

Example 2 In Vitro Synthesis of RNA Molecules with Modified NucleosidesMaterials and Experimental Methods

In Vitro-Transcribed RNA

Using in vitro transcription assays (MessageMachine and MegaScript kits;Ambion) the following long RNAs were generated by T7 RNA polymerase(RNAP) as described (Kariko et al, 1998, Phosphate-enhanced transfectionof cationic lipid-complexed mRNA and plasmid DNA. Biochim Biophys Acta1369, 320-334) (Note: the names of templates are indicated inparenthesis; the number in the name of the RNA specifies the length):RNA-1866 (Nde I-linearized pTEVluc) encodes firefly luciferase and a 50nt-long polyA-tail. RNA-1571 (Ssp I-linearized pSVren) encodes Renillaluciferase. RNA-730 (Hind III-linearized pT7T3D-MART-1) encodes thehuman melanoma antigen MART-1. RNA-713 (EcoR I-linearized pT7T3D-MART-1)corresponds to antisense sequence of MART-1, RNA-497 (Bgl II-linearizedpCMV-hTLR3) encodes a partial 5′ fragment of hTLR3. Sequences of the RNAmolecules are as follows:

RNA-1866: (SEQ ID No: 1)ggaauucucaacacaacauauacaaaacaaacgaaucucaagcaaucaagcauucuacuucuauugcagcaauuuaaaucauuucuuuuaaagcaaaagcaauuuucugaaaauuuucaccauuuacgaacgauagccauggaagacgccaaaaacauaaagaaaggcccggcgccauucuauccucuagaggauggaaccgcuggagagcaacugcauaaggcuaugaagagauacgcccugguuccuggaacaauugcuuuuacagaugcacauaucgaggugaacaucacguacgcggaauacuucgaaauguccguucgguuggcagaagcuaugaaacgauaugggcugaauacaaaucacagaaucgucguaugcagugaaaacucucuucaauucuuuaugccgguguugggcgcguuauuuaucggaguugcaguugcgcccgcgaacgacauuuauaaugaacgugaauugcucaacaguaugaacauuucgcagccuaccguaguguuuguuuccaaaaagggguugcaaaaaauuuugaacgugcaaaaaaaauuaccaauaauccagaaaauuauuaucauggauucuaaaacggauuaccagggauuucagucgauguacacguucgucacaucucaucuaccucccgguuuuaaugaauacgauuuuguaccagaguccuuugaucgugacaaaacaauugcacugauaaugaauuccucuggaucuacuggguuaccuaaggguguggcccuuccgcauagaacugccugcgucagauucucgcaugccagagauccuauuuuuggcaaucaaaucauuccggauacugcgauuuuaaguguuguuccauuccaucacgguuuuggaauguuuacuacacucggauauuugauauguggauuucgagucgucuuaauguauagauuugaagaagagcuguuuuuacgaucccuucaggauuacaaaauucaaagugcguugcuaguaccaacccuauuuucauucuucgccaaaagcacucugauugacaaauacgauuuaucuaauuuacacgaaauugcuucugggggcgcaccucuuucgaaagaagucggggaagcgguugcaaaacgcuuccaucuuccagggauacgacaaggauaugggcucacugagacuacaucagcuauucugauuacacccgagggggaugauaaaccgggcgcggucgguaaaguuguuccauuuuuugaagcgaagguuguggaucuggauaccgggaaaacgctgggcguuaaucagagaggcgaauuaugugucagaggaccuaugauuauguccgguuauguaaacaauccggaagcgaccaacgccuugauugacaaggauggauggcuacauucuggagacauagcuuacugggacgaagacgaacacuucuucauaguugaccgcuugaagucuuuaauuaaauacaaaggauaucagguggcccccgcugaauuggaaucgauauuguuacaacaccccaacaucuucgacgcgggcguggcaggucuucccgacgaugacgccggugaacuucccgccgccguuguuguuuuggagcacggaagacgaugacggaaaaagagaucguggauuacguggccagucaaguaacaaccgcgaaaaaguugcgcggaggaguuguguuuguggacgaaguaccgaaaggucuuaccggaaaacucgacgcaagaaaaaucagagagauccucauaaaggccaagaagggcggaaaguccaaauuguaaaauguaacucuagaggauccccaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaca. RNA-1571: (SEQ ID No: 2)ggcuagccaccaugacuucgaaaguuuaugauccagaacaaaggaaacggaugauaacugguccgcaguggugggcacagauguaaacaaaugaauguucuugauucauuuauuaauuauuaugauucagaaaaacaugcagaaaaugcuguuauuuuuuuacaugguaacgcggccucuucuuauuuauggcgacauguugugccacauauugagccaguagcgcgguguauuauaccagaccuuauugguaugggcaaaucaggcaaaucugguaaugguucuuauagguuacuugaucauuacaaauaucuuacugcaugguuugaacuucuuaauuuaccaaagaagaucauuuuugucggccaugauuggggugcuuguuuggcauuucauuauagcuaugagcaucaagauaagaucaaagcaauaguucacgcugaaaguguaguagaugugauugaaucaugggaugaauggccugauauugaagaagauauugcguugaucaaaucugaagaaggagaaaaaaugguuuuggagaauaacuucuucguggaaaccauguugccaucaaaaucaugagaaaguuagaaccagaagaauuugcagcauaucuugaaccauucaaagagaaaggugaaguucgucguccaacauuaucauggccucgugaaaucccguuaguaaaaggugguaaaccugacguuguacaaauuguuaggaauuauaaugcuuaucuacgugcaagugaugauuuaccaaaaauguuuauugaaucggacccaggauucuuuuccaaugcuauuguugaaggugccaagaaguuuccuaauacugaauuugucaaaguaaaaggucuucauuuuucgcaagaagaugcaccugaugaaaugggaaaauauaucaaaucguucguugagcgaguucucaaaaaugaacaaaugucgacgggggccccuaggaauuuuuuagggaagaucuggccuuccuacaagggaaggccagggaauuuucuucagagcagaccagagccaacagccccaccagaagagagcuucaggucugggguagagacaacaacucccccucagaagcaggagccgauagacaaggaacuguauccuuuaacuucccucagaucacucuuuggcaacgaccccucgucacaauaaagauaggggggcaacuaaagggaucggccgcuucgagcagacaugauaagauacauugaugaguuuggacaaaccacaacuagaaugcagugaaaaaaaugcuuuauuugugaaauuugugaugcuauugcuuuauuuguaaccauuauaagcugcaauaaacaaguuaacaacaacaauugcauucauuuuauguuucagguucagggggaggugugggagguuuuuuaaagcaaguaaaaccucuacaaaugugguaaaaucgauaaguuuaaacagauccagguggcacuuuucggggaaaugugcgcggaaccccuauuuguuuauuuuucuaaauacauucaaauauguauccgcucaugagacaauaacccugauaaaugcuucaauaau. RNA-730: (SEQ ID No: 3)gggaauuuggcccucgaggccaagaauucggcacgaggcacgcggccagccagcagacagaggacucucauuaaggaagguguccugugcccugacccuacaagaugccaagagaagaugcucacuucaucuaugguuaccccaagaaggggcacggccacucuuacaccacggcugaagaggccgcugggaucggcauccugacagugauccugggagucuuacugcucaucggcuguugguauuguagaagacgaaauggauacagagccuugauggauaaaagucuucauguuggcacucaaugugccuuaacaagaagaugcccacaagaaggguuugaucaucgggacagcaaagugucucuucaagagaaaaacugugaaccugugguucccaaugcuccaccugcuuaugagaaacucucugcagaacagucaccaccaccuuauucaccuuaagagccagcgagacaccugagacaugcugaaauuauuucucucacacuuuugcuugaauuuaauacagacaucuaauguucuccuuuggaaugguguaggaaaaaugcaagccaucucuaauaauaagucaguguuaaaauuuuaguagguccgcuagcaguacuaaucaugugaggaaaugaugagaaauauuaaauugggaaaacuccaucaauaaauguugcaaugcaugauaaaaaaaaaaaaaaaaaaaacugcggccgca.RNA-713 (SEQ ID No: 4)gggaauaagcuugcggccgcaguuuuuuuuuuuuuuuuuuuuaucaugcauugcaacauuuauugauggaguuuucccaauuuaauauuucucaucauuuccucacaugauuaguacugcuagcggaccuacuaaaauuuuaacacugacuuauuauuagagauggcuugcauuuuuccuacaccauuccaaaggagaacauuagaugucuguauaaauucaagcaaaagugugagagaaauaauuucagcaugucucaggugucucgcuggcucuuaaggugaauaaggugguggugacuguucugcagagaguuucucauaagcagguggagcauugggaaccacagguucacaguuuuucucuugaagagacacuuugcugucccgaugaucaaacccuucuugugggcaucuucuuguuaaggcacauugagugccaacaugaagacuuuuauccaucaaggcucuguauccauuucgucuucuacaauaccaacagccgaugagcaguaagacucccaggaucacugucaggaugccgaucccagcggccucuucagccgugguguaagaguggccgugccccuucuugggguaaccauagaugaagugagcaucuucucuuggcaucuuguagggucagggcacaggacaccuuccuuaaugagaguccucugucugcuggcuggccgcgugccucgugccgaauu. RNA-497: (SEQ ID No: 5)gggagacccaagcuggcuagcagucauccaacagaaucaugagacagacuuugccuuguaucuacuuuugggggggccuuuugcccuuugggaugcugugugcauccuccaccaccaagugcacuguuagccaugaaguugcugacugcagccaccugaaguugacucagguacccgaugaucuacccacaaacauaacaguguugaaccuuacccauaaucaacucagaagauuaccagccgccaacuucacaagguauagccagcuaacuagcuuggauguaggauuuaacaccaucucaaaacuggagccagaauugugccagaaacuucccauguuaaaaguuuugaaccuccagcacaaugagcuaucucaacuuucugauaaaaccuuugccuucugcacgaauuugacugaacuccaucucauguccaacucaauccagaaaauuaaaaauaaucccuuugucaagcagaagaauuuaaucacauua.

To obtain modified RNA, the transcription reaction was assembled withthe replacement of one (or two) of the basic NTPs with the correspondingtriphosphate-derivative(s) of the modified nucleotide 5-methylcytidine,5-methyluridine, 2-thiouridine, N⁶-methyladenosine or pseudouridine(TriLink, San Diego, Calif.). In each transcription reaction, all 4nucleotides or their derivatives were present at 7.5 millimolar (mM)concentration. In selected experiments, as indicated, 6 mM m7 GpppG capanalog (New England BioLabs, Beverly, Mass.) was also included to obtaincapped RNA. ORN5 and ORN6 were generated using DNA oligodeoxynucleotidetemplates and T7 RNAP (Silencer® siRNA construction kit, Ambion).

Results

To further test the effect of nucleoside modifications onimmunogenicity, an in vitro system was developed for producing RNAmolecules with pseudouridine or modified nucleosides. In vitrotranscription reactions were performed in which 1 or 2 of the 4nucleotide triphosphates (NTP) were substituted with a correspondingnucleoside-modified NTP. Several sets of RNA with different primarysequences ranging in length between 0.7-1.9 kb, and containing eithernone, 1 or 2 types of modified nucleosides were transcribed. ModifiedRNAs were indistinguishable from their non-modified counterparts intheir mobility in denaturing gel electrophoresis, showing that they wereintact and otherwise unmodified (FIG. 2A). This procedure workedefficiently with any of T7, SP6, and T3 phage polymerases, and thereforeis generalizable to a wide variety of RNA polymerases.

These findings provide a novel in vitro system for production of RNAmolecules with modified nucleosides.

Example 3 In Vitro-Transcribed RNA Stimulates Human TLR3, and NucleosideModifications Reduce the Immunogenicity of RNA Materials andExperimental Methods

Parental 293, 293-hTLR7 and 293-hTLR8 cells, all expressingTLR3-specific siRNA, and 293-hTLR9, TLR3-293 were seeded into 96-wellplates (5×10⁴ cells/well) and cultured without antibiotics. On thesubsequent day, the cells were exposed to R-848 or RNA complexed toLipofectin® (Invitrogen) as described (Kariko et al, 1998, ibid). RNAwas removed after one hour (h), and cells were further incubated incomplete medium for 7 h. Supernatants were collected for IL-8measurement.

Results

To determine whether modification of nucleosides influences theRNA-mediated activation of TLRs, human embryonic kidney 293 cells werestably transformed to express human TLR3. The cell lines were treatedwith Lipofectin®-complexed RNA, and TLR activation was monitored asindicated by interleukin (IL)-8 release. Several different RNA moleculeswere tested. Unmodified, in vitro-transcribed RNA elicited a high levelof IL-8 secretion. RNA containing m6A or s2U nucleoside modifications,but contrast, did not induce detectable IL-8 secretion (FIG. 2B). Theother nucleoside modifications tested (i.e. m5C, m5U, Ψ, and m5C/Ψ) hada smaller suppressive effect on TLR3 stimulation (FIG. 2B). “Ψ” refersto pseudouridine.

Thus, nucleoside modifications such as m⁶A s²U, m⁵C, m⁵U, Ψ, reduce theimmunogenicity of RNA as mediated by TLR3 signaling.

Example 4 In Vitro-Transcribed RNA Stimulates Human TLR7 and TLR8, andNucleoside Modifications Reduce the Immunogenicity of RNA

To test the possibility that 293 express endogenous TLR3 that interferewith assessing effects of RNA on specific TLR receptors, expression ofendogenous TLR3 was eliminated from the 293-TLR8 cell line by stablytransfecting the cells with a plasmid expressing TLR3-specific shorthairpin (sh)RNA (also known as siRNA). This cell line was used forfurther study, since it did not respond to poly(I):(C), LPS, andCpG-containing oligodeoxynucleotides (ODNs), indicating the absence ofTLR3, TLR4 and TLR9, but did respond to R-848, the cognate ligand ofhuman TLR8 (FIG. 2B). When the 293-hTLR8 cells expressing TLR3-targetedshRNA (293-hTLR8 shRNA-TLR3 cells) were transfected with invitro-transcribed RNA, they secreted large amounts of IL-8. By contrast,RNA containing most of the nucleoside modifications (m⁵C, m⁵U, Ψ, andm⁵C/T, s²U) eliminated stimulation (no more IL-8 production than thenegative control, i.e. empty vector). m6A modification had a variableeffect, in some cases eliminating and in other cases reducing IL-8release (FIG. 2B).

The results of this Example and the previous Example show that (a) RNAwith natural phosphodiester inter-nucleotide linkages (e.g. invitro-transcribed RNA) stimulates human TLR3, TLR7 and TLR8; and (b)nucleoside modifications such as m6A, m5C, m5U, s2U and Ψ, alone and incombination, reduce the immunogenicity of RNA as mediated by TLR3, TLR7and TLR8 signaling. In addition, these results provide a novel systemfor studying signaling by specific TLR receptors.

Example 5 Nucleoside Modifications Reduce the Immunogenicity of RNA asMediated by TLR7 and TLR8 Signaling

The next set of experiments tested the ability of RNA isolated fromnatural sources to stimulate TLR3, TLR7 and TLR8. RNA from differentmammalian species were transfected into the TLR3, TLR7 andTLR8-expressing 293 cell lines described in the previous Example. Noneof the mammalian RNA samples induced IL-8 secretion above the level ofthe negative control. By contrast, bacterial total RNA obtained from twodifferent E. coli sources induced robust IL-8 secretion in cellstransfected with TLR3, TLR7 and TLR8, but not TLR9 (FIG. 2C). NeitherLPS nor unmethylated DNA (CpG ODN) (the potential contaminants inbacterial RNA isolates) activated the tested TLR3, TLR7 or TLR8.Mitochondrial RNA isolated from human platelets stimulated human TLR8,but not TLR3 or TLR7.

These results demonstrate that unmodified in vitro-transcribed andbacterial RNA are activators of TLR3, TLR7 and TLR8, and mitochondrialRNA stimulates TLR8. In addition, these results confirm the finding thatnucleoside modification of RNA decreases its ability to stimulate TLR3,TLR7 and TLR8.

Example 6 Nucleoside Modifications Reduce the Capacity of RNA to InduceCytokine Secretion and Activation Marker Expression by DC Materials andExperimental Methods

DC Stimulation Assays

After 20 h of incubation with RNA, DCs were stained withCD83-phycoerythrin mAb (Research Diagnostics Inc, Flanders, N.J.),HLA-DR-Cy5PE, and CD80 or CD86-fluorescein isothiocyanate mAb andanalyzed on a FACScalibur® flow cytometer using CellQuest® software (BDBiosciences). Cell culture supernatants were harvested at the end of a20 h incubation and subjected to cytokine ELISA. The levels of IL-12(p70) (BD Biosciences Pharmingen, San Diego, Calif.), IFN-α, and TNF-α(Biosource International, Camarillo, Calif.) were measured insupernatants by ELISA. Cultures were performed in triplicate orquadruplicate, and each sample was measured in duplicate.

Results

The next experiments tested the ability of RNA containing modified orunmodified nucleosides to stimulate cytokine-generated MDDC. Nucleosidemodifications reproducibly diminished the ability of RNA to induce TNF-αand IL-12 secretion by both GM-CSF/IL-4-generated MDDC and(GM-CSF)/IFN-α-generated MDDC, in most cases to levels no greater thanthe negative control (FIGS. 3A and B). Results were similar when othersets of RNA with the same base modifications but different primarysequences and lengths were tested, or when the RNA was further modifiedby adding a 5′ cap structure and/or 3′-end polyA-tail or by removing the5′ triphosphate moiety. RNAs of different length and sequence inducedvarying amounts of TNF-α from DC, typically less than a two-folddifference (FIG. 3C).

Next, the assay was performed on primary DC1 and DC2. Primary monocytoid(DC1, BDCA1⁺) and plasmacytoid (DC2, BDCA4⁺) DC were purified fromperipheral blood. Both cell types produced TNF-α when exposed to R-848,but only DC1 responded to poly(I):(C), at a very low level, indicatingan absence of TLR3 activity in DC2. Transfection of in vitro transcriptsinduced TNF-α secretion in both DC1 and DC2, while m5U, Ψ ors2U-modified transcripts were not stimulatory (FIG. 3D). In contrast tothe cytokine-generated DC, m5C and m6A modification of RNA did notdecrease its stimulatory capacity in the primary DC1 and DC2.Transcripts with m6A/Ψ double modification were non-stimulatory, while amixture of RNA molecules with single type of modification (m6A+Ψ) was apotent cytokine inducer. Thus, uridine modification exerted a dominantsuppressive effect on an RNA molecule in cis in primary DC. Theseresults were consistent among all donors tested.

These findings show that in vitro-transcribed RNA stimulates cytokineproduction by DC. In addition, since DC2 do not express TLR3 or TLR8,and m5C and m6A modification of RNA decreased its stimulatory capacityof TLR7, these findings show that primary DC have an additional RNAsignaling entity that recognizes m5- and m6A-modified RNA and whosesignaling is inhibited by modification of U residues.

As additional immunogenicity indicators, cell surface expression ofCD80, CD83, CD86 and MHC class II molecules, and secretion of TNF-α weremeasured by FACS analysis of MDDC treated with RNA-1571 and its modifiedversions. Modification of RNA with pseudouridine and modifiednucleosides (m5C, m6A, s2U and m6A/Ψ) decreased these markers (FIG. 4),confirming the previous findings.

In summary, RNA's capacity to induce DCs to mature and secrete cytokinesdepends on the subtype of DC as well as on the characteristics ofnucleoside modification present in the RNA. An increasing amount ofmodification decreases the immunogenicity of RNA.

Example 7 Suppression of RNA-Mediated Immune Stimulation is Proportionalto the Number of Modified Nucleosides Present in RNA Materials andExperimental Methods

Human DC

For cytokine-generated DC, monocytes were purified from PBMC bydiscontinuous Percoll gradient centrifugation. The low density fraction(monocyte enriched) was depleted of B, T, and, NK cells using magneticbeads (Dynal, Lake Success, N.Y.) specific for CD2, CD16, CD19, andCD56, yielding highly purified monocytes as determined by flow cytometryusing anti-CD14 (>95%) or anti-CD11c (>98%) mAb.

To generate immature DC, purified monocytes were cultured in AIM Vserum-free medium (Life Technologies), supplemented with GM-CSF (50ng/ml)+IL-4 (100 ng/ml) (R & D Systems, Minneapolis, Minn.) in AIM Vmedium (Invitrogen) for the generation of monocyte-derived DC (MDDC) asdescribed (Weissman, D et al, 2000. J Immunol 165: 4710-4717). DC werealso generated by treatment with GM-CSF (50 ng/ml)+IFN-α (1,000 U/ml) (R& D Systems) to obtain IFN-α MDDC (Santini et al., 2000. Type Iinterferon as a powerful adjuvant for monocyte-derived dendritic celldevelopment and activity in vitro and in Hu-PBL-SCID mice. J Exp Med191: 1777-178).

Primary myeloid and plasmacytoid DCs (DC1 and DC2) were obtained fromperipheral blood using BDCA-1 and BDCA-4 cell isolation kits (MiltenyiBiotec Auburn, Calif.), respectively.

Results

Most of the nucleoside-modified RNA utilized thus far contained one typeof modification occurring in approximately 25% of the total nucleotidesin the RNA (e.g. all the uridine bases). To define the minimal frequencyof particular modified nucleosides that is sufficient to reduceimmunogenicity under the conditions utilized herein, RNA molecules withlimited numbers of modified nucleosides were generated. In the first setof experiments, RNA was transcribed in vitro in the presence of varyingratios of m6A, Ψ or m5C to their corresponding unmodified NTPs. Theamount of incorporation of modified nucleoside phosphates into RNA wasexpected to be proportional to the ratio contained in the transcriptionreaction, since RNA yields obtained with T7 RNAP showed the enzymeutilizes NTPs of m6A, Ψ or m5C almost as efficiently as the basic NTPs.To confirm this expectation, RNA transcribed in the presence of UTP:Ψ ina 50:50 ratio was digested and found to contain UMP and Ψ in a nearly50:50 ratio (FIG. 5A).

RNA molecules with increasing modified nucleoside content weretransfected into MDDC, and TNF-α secretion was assessed. Eachmodification (m6A, Ψ and m5C) inhibited TNF-α secretion proportionallyto the fraction of modified bases. Even the smallest amounts of modifiedbases tested (0.2-0.4%, corresponding to 3-6 modified nucleosides per1571 nt molecule), was sufficient to measurably inhibit cytokinesecretion (FIG. 5B). RNA with of 1.7-3.2% modified nucleoside levels(14-29 modifications per molecule) exhibited a 50% reduction ininduction of TNF-α expression. In TLR-expressing 293 cells, a higherpercentage (2.5%) of modified nucleoside content was required to inhibitRNA-mediated signaling events.

Thus, pseudouridine and modified nucleosides reduce the immunogenicityof RNA molecules, even when present as a small fraction of the residues.

In additional experiments, 21-mer oligoribonucleotides (ORN) withphosphodiester inter-nucleotide linkages were synthesized whereinmodified nucleosides (m5C, Ψ or 2′-O-methyl-U [Um]) were substituted ina particular position (FIG. 6A). While the unmodified ORN induced TNF-αsecretion, this effect was abolished by the presence of a singlenucleoside modification (FIG. 6B). Similar results were obtained withTLR-7 and TLR-8-transformed 293 cells expressing TLR3-targeted siRNA.

The above results were confirmed by measuring TNF-α mRNA levels in MDDCby Northern blot assay, using both the above 21-mer ORN(ORN1) and 31-merin vitro-synthesized transcripts (ORN5 and ORN6). To amplify the signal,cycloheximide, which blocks degradation of selected mRNAs, was added tosome samples, as indicated in the Figure. The unmodified ODN increasedTNF-α mRNA levels, while ORNs containing a single modified nucleosidewere significantly less stimulatory; ORN2-Um exhibited the greatestdecrease TNF-α production (FIG. 6C).

Similar results were observed in mouse macrophage-like RAW cells and inhuman DC.

In summary, each of the modifications tested (m6A, m5C, m5U, s2U, Ψ and2′-O-methyl) suppressed RNA-mediated immune stimulation, even whenpresent as a small fraction of the residues. Further suppression wasobserved when the proportion of modified nucleosides was increased.

Example 8 Pseudouridine-Modification of RNA Reduces its ImmunogenicityIn Vivo

To determine the effect of pseudouridine modification on immunogenicityof RNA in vivo, 0.25 μg RNA) was complexed to Lipofectin® and injectedintra-tracheally into mice, mice were bled 24 h later, and circulatinglevels of TNF-α and IFN-α were assayed from serum samples. Capped,pseudouridine-modified mRNA induced significantly less TNF-α and IFN-αmRNA than was elicited by unmodified mRNA (FIG. 7A-B).

These results provide further evidence that pseudouridine-modified mRNAis significantly less immunogenic in vivo than unmodified RNA.

Example 9 Pseudouridine-Containing RNA Exhibits Decreased Ability toActivate PRK Materials and Experimental Methods

PKR Phosphorylation Assays

Aliquots of active PKR agarose (Upstate) were incubated in the presenceof magnesium/ATP coctail (Upstate), kinase buffer and [gamma³²P] ATP mixand RNA molecules for 30 min at 30° C. Unmodified RNA and RNA withnucleoside modification (m5C, pseudouridine, m6A, m5U) and dsRNA weretested. Human recombinant eIF2a (BioSource) was added, and samples werefurther incubated for 5 min, 30° C. Reactions were stopped by addingNuPage LDS sample buffer with reducing reagent (Invitrogen), denaturedfor 10 min, 70° C., and analyzed on 10% PAGE. Gels were dried andexposed to film. Heparin (1 U/μl), a PKR activator, was used as positivecontrol.

Results

To determine whether pseudouridine-containing mRNA activatesdsRNA-dependent protein kinase (PKR), in vitro phosphorylation assayswere performed using recombinant human PKR and its substrate, eIF2α(eukaryotic initiation factor 2 alpha) in the presence of capped,renilla-encoding mRNA (0.5 and 0.05 ng/μl). mRNA containingpseudouridine (Ψ) did not activate PKR, as detected by lack of bothself-phosphorylation of PKR and phosphorylation of eIF2a, while RNAwithout nucleoside modification and mRNA with m⁵C modification activatedPKR (FIG. 8). Thus, pseudouridine modification decreases RNAimmunogenicity.

Example 10 Enhanced Translation of Proteins from Pseudouridine andm⁵C-Containing RNA In Vitro Materials and Experimental Methods

In Vitro Translation of mRNA in Rabbit Reticulocyte Lysate

In vitro-translation was performed in rabbit reticulocyte lysate(Promega, Madison Wis.). A 9-μl aliquot of the lysate was supplementedwith 1 μl (1 μg) mRNA and incubated for 60 min at 30° C. One μl aliquotwas removed for analysis using firefly and renilla assay systems(Promega, Madison Wis.), and a LUMAT LB 950 luminometer (Berthold/EG&GWallac, Gaithersburg, Md.) with a 10 sec measuring time.

Results

To determine the effect of pseudouridine modification on RNA translationefficiency in vitro, (0.1 μg/μl) uncapped mRNA modified withpseudouridine encoding firefly luciferase was incubated in rabbitreticulocyte lysate for 1 h at 30° C., and luciferase activity wasdetermined. mRNA containing pseudouridine was translated more than2-fold more efficiently than RNA without pseudouridine in rabbitreticulocyte lysates, but not in wheat extract or E. coli lysate (FIG.9), showing that pseudouridine modification increases RNA translationefficiency. Similar results were obtained with m⁵C-modified RNA. When apolyA tail was added to pseudouridine-containing mRNA, a further 10-foldincrease in translation efficiency was observed. (Example 10).

Thus, pseudouridine and m⁵C modification increases RNA translationefficiency, and addition of a polyA tail to pseudouridine-containingmRNA further increases translation efficiency.

Example 11 Enhanced Translation of Proteins fromPseudouridine-Containing RNA in Cultured Cells Materials andExperimental Methods

Translation Assays in Cells

Plates with 96 wells were seeded with 5×10⁴ cells per well 1 day beforetransfection. Lipofectin®-mRNA complexes were assembled and addeddirectly to the cell monolayers after removing the culture medium (0.2μg mRNA-0.8 μg lipofectin in 50 μA per well). Cells were incubated withthe transfection mixture for 1 h at 37° C., 5% CO₂ incubator, then themixture was replaced with fresh, pre-warmed medium containing 10% FCS,then cells were analyzed as described in the previous Example.

Results

To determine the effect of pseudouridine modification on RNA translationin cultured cells, 293 cells were transfected with in vitro-transcribed,nucleoside-modified, capped mRNA encoding the reporter protein renilla.Cells were lysed 3 h after initiation of transfection, and levels ofrenilla were measured by enzymatic assays. In 293 cells, pseudouridine-and m5C-modified DNA were translated almost 10 times and 4 times moreefficiently, respectively, than unmodified mRNA (FIG. 10A).

Next, the experiment was performed with primary, bone marrow-derivedmouse DC, in this case lysing the cells 3 h and 8 h after transfection.RNA containing the pseudouridine modification was translated 15-30 timesmore efficiently than unmodified RNA (FIG. 10B).

Similar expression results were obtained using human DC and otherprimary cells and established cell lines, including CHO and mousemacrophage-like RAW cells. In all cell types, pseudouridine modificationproduced the greatest enhancement of the modifications tested.

Thus, pseudouridine modification increased RNA translation efficiency inall cell types tested, including different types of both professionalantigen-presenting cells and non-professional antigen-presenting cells,providing further evidence that pseudouridine modification increases theefficiency of RNA translation.

Example 12 5′ and 3′ Elements Further Enhance the Translation of ψmRNAin Mammalian Cells

To test the effect of additional RNA structural elements on enhancementof translation by pseudouridine modification, a set of fireflyluciferase-encoding ψmRNAs were synthesized that contained combinationsof the following modifications: 1) a unique 5′ untranslated sequence(TEV, a cap independent translational enhancer), 2) cap and 3)polyA-tail. The ability of these modifications to enhance translation ofψmRNA or conventional mRNA was assessed (FIG. 11A). These structuralelements additively enhanced translational efficiency of bothconventional and ψmRNA, with ψmRNA exhibiting greater protein productionfrom all constructs.

Ability of protein expression from the most efficient firefly luciferaseψmRNA construct, capTEVlucA50 (containing TEV, cap, and an extendedpoly(A) tail) was next examined over 24 hours in 293 cells (FIG. 11B).ψmRNA produced more protein at every time point tested and conferredmore persistent luciferase expression than equivalent conventional mRNAconstructs, showing that ψ-modifications stabilize mRNA.

To test whether ψ-modification of mRNA improved translation efficiencyin mammalian cells in situ, caplacZ-ψmRNA constructs with or withoutextended polyA-tails (A_(n)) and encoding β-galactosidase (lacZ) weregenerated and used to transfect 293 cells. 24 h after mRNA delivery,significant increases in β-galactosidase levels were detected by X-galvisualization, in both caplacZ and caplacZ-A_(n), compared to thecorresponding control (conventional) transcripts (FIG. 11C). This trendwas observed when either the number of cells expressing detectablelevels of β-galactosidase or the signal magnitude in individual cellswas analyzed.

Example 13 Enhanced Translation of Proteins fromPseudouridine-Containing RNA In Vivo Materials and Experimental Methods

Intracerebral RNA Injections

All animal procedures were in accordance with the NIH Guide for Care andUse of Laboratory Animals and approved by the Institutional Animal Careand Use Committee. Male Wistar rats (Charles River Laboratories,Wilmington, Mass.) were anesthetized by intraperitoneal injection ofsodium pentobarbital (60 mg/kg body weight). Heads were placed in astereotaxic frame, and eight evenly spaced 1.5 mm diameter burr holeswere made bilaterally [coordinates relative to bregma:anterior/posterior +3, 0, −3, −6 mm; lateral±2.5 mm] leaving the duraintact. Intracerebral injections were made using a 25 μl syringe(Hamilton, Reno, Nev.) with a 30 gauge, 1 inch sterile needle (BecktonDickinson Labware, Franklin Lakes, N.J.) which was fixed to a largeprobe holder and stereotactic arm. To avoid air space in the syringe,the needle hub was filled with 55 μl complex before the needle wasattached, and the remainder of the sample was drawn through the needle.Injection depth (2 mm) was determined relative to the surface of thedura, and 4 μl complex (32 ng mRNA) was administered in a single, rapidbolus infusion. 3 hours (h) later, rats were euthanized with halothane,and brains were removed into chilled phosphate buffered saline.

Injection of RNA into Mouse Tail Vein

Tail veins of female BALB/c mice (Charles River Laboratories) wereinjected (bolus) with 60 μl Lipofectin®-complexed RNA (0.26 μg). Organswere removed and homogenized in luciferase or Renilla lysis buffer inmicrocentrifuge tubes using a pestle. Homogenates were centrifuged, andsupernatants were analyzed for activity.

Delivery of RNA to the Lung

Female BALB/c mice were anaesthetized using ketamine (100 mg/kg) andxylasine (20 mg/kg). Small incisions were made in the skin adjacent tothe trachea. When the trachea was exposed, 50 μl ofLipofectin®-complexed RNA (0.2 μg) was instilled into the tracheatowards the lung. Incisions were closed, and animals allowed to recover.3 hours after RNA delivery, mice were sacrificed by cervical dislocationand lungs were removed, homogenized in luciferase or Renilla lysisbuffer (250 μA), and assayed for activity. In a different set ofanimals, blood samples (100 μl/animal) were collected from tail veins,clotted, and centrifuged. Serum fractions were used to determine levelsof TNF and IFNα by ELISA as described in the Examples above, usingmouse-specific antibodies.

Results

To determine the effect of pseudouridine modification on RNA translationin vivo, each hemisphere of rat brain cortexes was injected with eithercapped, renilla-encoding pseudouridine-modified RNA or unmodified RNA,and RNA translation was measured. Pseudouridine-modified RNA wastranslated significantly more efficiently than unmodified RNA (FIG.12A).

Next, expression studies were performed in mice. Fireflyluciferase-encoding mRNAs because no endogenous mammalian enzymeinterferes with its detection. Transcripts (unmodified and ψmRNA) wereconstructed with cap, TEV (capTEVA₅₀) and extended (˜200 nt) poly(A)tails. 0.25 μg RNA Lipofectin®-complexed was injected into mice(intravenous (i.v.) tail vein). A range of organs were surveyed forluciferase activity to determine the optimum measurement site.Administration of 0.3 μg capTEVlucAn ψmRNA induced high luciferaseexpression in spleen and moderate expression in bone marrow, but littleexpression in lung, liver, heart, kidney or brain (FIG. 12B). Insubsequent studies, spleens were studied.

Translation efficiencies of conventional and ψmRNA (0.015 mg/kg; 0.3μg/animal given intravenously) were next compared in time courseexperiments. Luciferase activity was readily detectable at 1 h, peakedat 4 h and declined by 24 h following administration of eitherconventional or ψmRNA, but at all times was substantially greater inanimals given ψmRNA (FIG. 12C, left panel). By 24 h, only animalsinjected with ψmRNA demonstrated detectable splenic luciferase activity(4-fold above background). A similar relative pattern of expression(between modified and unmodified mRNA) was obtained when mRNAs encodingRenilla luciferase (capRen with or without ψ modifications) wereinjected into the animals instead of firefly luciferase, or whenisolated mouse splenocytes were exposed to mRNA in culture.

In the next experiment, 0.25 μg mRNA-Lipofectin® was delivered to mouselungs by intra-tracheal injection. Capped, pseudouridine-modified RNAwas translated more efficiently than capped RNA without pseudouridinemodification (FIG. 12D).

Thus, pseudouridine modification increases RNA translation efficiency invitro, in cultured cells, and in vivo—in multiple animal models and bymultiple routes of administration, showing its widespread application asa means of increasing the efficiency of RNA translation.

Example 14 Pseudouridine Modification Enhances RNA Stability In Vivo

Northern analyses of splenic RNA at 1 and 4 h post injection in theanimals from the previous Example revealed that the administered mRNAs,in their intact and partially degraded forms, were readily detectable(FIG. 12C, right panel). By contrast, at 24 h, unmodified capTEVlucAnmRNA was below the level of detection, while capTEVlucAn ψmRNA, thoughpartially degraded, was still clearly detectable. Thus, ψmRNA is morestably preserved in vivo than control mRNA.

To test whether in vivo protein production is quantitatively dependenton the concentration of intravenously-delivered mRNA, mRNAs wereadministered to mice at 0.015-0.150 mg/kg (0.3-3.0 μg capTEVlucAn peranimal) and spleens were analyzed 6 hours later as described above.Luciferase expression correlated quantitatively with the amount ofinjected RNA (FIG. 13) and at each concentration.

These findings confirm the results of Example 12, demonstrating thatψmRNA is more stable than unmodified RNA.

Further immunogenicity of ψ-mRNA was less than unmodified RNA, asdescribed hereinabove (FIG. 7 and FIG. 12C, right panel).

To summarize Examples 13-14, the 3 advantages of ψ-mRNA compared withconventional mRNA (enhanced translation, increased stability and reducedimmunogenicity) observed in vitro are also observed in vivo.

Example 15 ψmRNA Delivered Via the Respiratory Tract Behaves Similarlyto Intravenously Administered mRNA

To test the ability of ψmRNA to be delivered by inhalation, Lipofectin®-or PEI-complexed mRNAs encoding firefly luciferase were delivered tomice by the intratracheal route, wherein a needle was placed into thetrachea and mRNA solution sprayed into the lungs. Similar to intravenousdelivery, significantly greater luciferase expression was observed withψmRNA compared to unmodified mRNA (FIG. 14), although significantly lessprotein was produced with the intratracheal as compared to theintravenous routes. Unmodified mRNA administered by the intratrachealroute was associated with significantly higher concentrations ofinflammatory cytokines (IFN-α and TNF-α) compared with vehicle controls,while ψmRNA was not (FIG. 15).

Thus, ψmRNA can be delivered by inhalation without activating the innateimmune response.

Example 16 Delivery of EPO-ψmRNA to 293 Cells

ψmRNA was generated from a plasmid containing the human EPO cDNA. When0.25 μg of EPO-ψmRNA was transfected into 10⁶ cultured 293 cells,greater than 600 mU/ml of EPO protein was produced. Thus, modified RNAmolecules of the present invention are efficacious at deliveringrecombinant proteins to cells.

Example 17 Preparation of Improved EPO-Encoding mRNA ConstructsMaterials and Experimental Methods

The EPO coding sequence is cloned using restriction enzyme techniques togenerate 2 new plasmids, pTEV-EPO and pT7TS-EPO, that are used astemplates for EPO-ψmRNA production. EPO-ψmRNAs willare produced fromthese templates by in vitro transcription (MessageMachine® andMegaScript® kits; Ambion) using T7 RNA polymerase (RNAP), incorporatingnucleosides at equimolar (7.5 mM) concentrations. To incorporate thenucleoside-modifications, ψ triphosphate (TriLink, San Diego, Calif.)replaces UTP in the transcription reaction. To ensure capping of theψmRNA, a non-reversible cap-analog, 6 mM 3′-O-Me-m7 GpppG (New EnglandBioLabs, Beverly, Mass.) is also included. The ψmRNAs are poly(A)-tailedin a reaction of ˜1.5 μg/μl RNA, 5 mM ATP, and 60 U/μ1 yeast poly(A)polymerase (USB, Cleveland, Ohio) mixed at 30° C. for 3 to 24 h. Qualityof ψmRNAs is assessed by denaturing agarose gel electrophoresis. Assaysfor LPS in mRNA preparations using the Limulus Amebocyte Lysate gel clotassay with a sensitivity of 3 pg/ml are also performed.

Results

The proximal 3′-untranslated region (3′UTR) of EPO-ψmRNA preserves a ˜90nt-long pyrimidine-rich stabilizing element from the nascent EPO mRNA,which stabilizes EPO mRNA by specific association with a ubiquitousprotein, erythropoietin mRNA-binding protein (ERBP). To maximize thestability of EPO-ψmRNA, 2 alterations are incorporated into the EPOplasmid to improve the stability and translational efficiency of thetranscribed mRNA: 1) A 5′UTR sequence of the tobacco etch virus (TEV) isincorporated upstream of the EPO coding sequence to generate pTEV-EPO.2) A plasmid, pT7TS-EPO, is generated, wherein the EPO cDNA is flankedby sequences corresponding to β-globin 5′ and 3′UTRs.

In addition, the length of the poly(A) tail during the production ofψmRNA from these plasmid templates is extended, by increasing theincubation period of the poly(A) polymerase reaction. The longer poly(A)tail diminishes the rate at which ψmRNA degrades during translation.

These improvements result in enhanced translation efficiency in vivo,thus minimizing the therapeutic dose of the final product.

Example 18 In Vitro Analysis of Protein Production from EPO mRNAConstructs Materials and Experimental Methods

Preparation of Mammalian Cells.

Human embryonic kidney 293 cells (ATCC) are propagated in DMEMsupplemented with glutamine (Invitrogen) and 10% FCS (Hyclone, Ogden,Utah) (complete medium). Leukopheresis samples are obtained fromHIV-uninfected volunteers through an IRB-approved protocol. DCs areproduced as described above and cultured with GM-CSF (50 ng/ml)+IL-4(100 ng/ml) (R & D Systems) in AIM V Medium® (Invitrogen).

Murine spleen cells and DC are obtained by published procedures.Briefly, spleens from BALB/c mice are aseptically removed and mincedwith forceps in complete medium. Tissue fragments are sedimented bygravity and the single cell suspension washed and lysed with AKC lysisbuffer (Sigma). Murine DCs are derived from bone marrow cells collectedfrom femurs and tibia of 6-9-week-old BALB/c mice. Cells are cultured inDMEM containing 10% FCS (Invitrogen) and 50 ng/ml muGM-CSF (R&D) andused on day 7.

Transfection of Cells and Detection of EPO and Pro-InflammatoryCytokines

Transfections are performed with Lipofectin in the presence of phosphatebuffer, an effective delivery method for splenic and in vitro cellexpression. EPO-ψmRNA (0.25 μg/well; 100,000 cells) is added to eachcell type in triplicate for 1 hour, and supernatant replaced with freshmedium. 24 hours later, supernatant is collected for ELISA measurementof EPO, IFN-α or β, and TNF-α.

Results

To evaluate the impact of unique UTRs on enhancement of ψmRNAtranslational efficiency, EPO-ψmRNA containing, or not containing, eachimprovement (5′ TEV element, β-globin 5′ and 3′UTRs) with long poly(A)tails are tested for in vitro protein production and in vitro immuneactivation, with. EPO conventional-nucleoside mRNA used as controls.Efficiency of protein production from each mRNA is assessed in mammaliancell lines, (HEK293, CHO), human and murine primary DCs, and spleencells for each mRNA. Measurement of total EPO produced in all cell typesand immunogenicity (supernatant-associated proinflammatory cytokines) inprimary cells is evaluated. The mRNA construct that demonstrates theoptimum combination of high EPO production (in 1 or more cell types) andlow cytokine elicitation is used in subsequent studies. Improvements in5′ and 3′UTRs of EPO-ψmRNA and longer poly(A) tails result in anestimated 2-10-fold enhancement in translation efficiency, with noincrease in immunogenicity.

Example 19 Characterization of EPO Production and Biological Response toEPO-ψmRNA In Vivo Materials and Experimental Methods

Administration of EPO-ψmRNA to Mice.

All animal studies described herein are performed in accordance with theNIH Guide for Care and Use of Laboratory Animals and approved by theInstitutional Animal Care and Use Committee of the University ofPennsylvania. Female BALB/c mice (n=5 per experimental condition; 6weeks, 18-23g; Charles River Laboratories) are anesthetized using 3.5%halothane in a mixture of N₂O and O₂ (70:30), then halothane reduced to1% and anesthesia maintained using a nose mask. Animal body temperaturesare maintained throughout the procedure using a 37° C. warmed heatingpad. EPO-ψmRNA-lipofectin complexes (constructed by mixing varyingamounts of nucleic acid with 1 μl lipofectin in 60 μl final volume areinjected into the lateral tail vein. Blood samples are collected 3 timesa day for 3 days post mRNA injection during the time-course study, at 1optimal time point in dose-response studies, and daily from days 2-6 instudies for reticulocytosis.

Determination of Reticulocytes by Flow Cytometry.

Whole blood samples are stained using Retic-COUNT reagent (BDDiagnostics) and data events acquired on a FACScan flow cytometer. Redblood cells (RBCs) are selected by forward and side scatter propertiesand analyzed for uptake of Thiazole Orange. Cells stained withRetic-COUNT reagent are detected by fluorescence and reticulocytesexpressed as the percentage of total RBC. At least 50,000 events arecounted per sample.

Results

To optimize production of biologically functional human EPO protein(hEPO) in response to EPO-encoding mRNA, the following studies areperformed:

Time Course of EPO Production After a Single Injection of EPO-ψmRNA.

Following intravenous administration of 1 μg PO-ψmRNA, hEPO is measuredserially from 1-96 h after EPO-ψmRNA administration by ELISA, todetermined the half-life of EPO protein in the serum will be determined.This half-life is a product of both the half-life of EPO protein and thefunctional half-life of the EPO-ψmRNA. The resulting optimal time pointfor measuring EPO protein after EPO-ψmRNA administration is utilized insubsequent studies.

Dose-Response of EPO Production After a Single Injection of EPO-ψmRNA.

To determine the correlation between the amount of EPO protein producedand the amount of EPO-ψmRNA administered, increasing concentrations ofEPO-ψmRNA (0.01 to 1 μg/animal) are administered and EPO will bemeasured at the optimal time point.

Relationship Between hEPO Production and Reticulocytosis.

To measure the effect of EPO-ψmRNA on a biological correlate of EPOactivity, flow cytometry is used to determine reticulocyte frequency inblood). Flow cytometry has a coefficient of variation of <3%. Micereceive a single dose of EPO-ψmRNA, and blood is collected from micedaily from days 2-6. The relationship between EPO-ψmRNA dose andreticulocyte frequency is then evaluated at the time point of maximalreticulocytosis. The dose of EPO-ψmRNA that leads to at least a 5%increase in reticulocyte count is used in subsequent studies. Serum hEPOconcentrations in mice of an estimated 50 mU/ml and/or an increase inreticulocyte frequency of an estimated 5% are obtained.

Example 20 Measuring Immune Responses to EPO-ψmRNA In Vivo Materials andExperimental Methods

Detection of Cytokines in Plasma.

Serum samples obtained from blood collected at different times duringand after 7 daily lipofectin-complexed mRNA administrations are analyzedfor mouse IFN-α, TNF-α, and IL-12 using ELISA kits.

Northern Blot Analysis.

Aliquots (2.0 μg) of RNA samples isolated from spleen are separated bydenaturing 1.4% agarose gel electrophoresis, transferred to chargedmembranes (Schleicher and Schuell) and hybridized in MiracleHyb®(Stratagene). Membranes are probed for TNF-α, down-stream IFN signalingmolecules (e.g. IRF7, IL-12 p35 and p40, and GAPDH) and other markers ofimmune activation. Specificity of all probes is confirmed by sequencing.To probe the membranes, 50 ng of DNA is labeled using Redivue[α-³²P]dCTP® (Amersham) with a random prime labeling kit (Roche). Hybridizedmembranes are exposed to Kodak BioMax MS film using an MS intensifierscreen at −70° C.

Histopathology.

Spleens from EPO-ψmRNA-treated and positive and negative control-treatedmice are harvested, fixed, sectioned, stained with hematoxylin and eosinand examined by a veterinary pathologist for signs of immune activation.

Results

To confirm the reduced immunogenicity of RNA molecules of the presentinvention, mice (n=5) receive daily doses of EPO-ψmRNA for 7 days, thenare evaluated for immune-mediated adverse events, as indicated by serumcytokine concentrations, splenic expression of mRNAs encodinginflammatory proteins, and pathologic examination. Maximum administereddoses are 3 μg or 5× the effective single dose as determined above.Unmodified mRNA and Lipofectin® alone are used as positive and negativecontrols, respectively.

These studies confirm the reduced immunogenicity of RNA molecules of thepresent invention.

Example 21 Further Improvement of EPO-ψmRNA Delivery Methods

Nanoparticle Complexing.

Polymer and ψmRNA solutions are mixed to form complexes. Variousformulation conditions are tested and optimized: (1) sub-22 nmpolyethylenimine (PEI)/mRNA complexes are made by addition of 25 volumesof mRNA to 1 volume of PEI in water with no mixing for 15 minutes. (2)The rod-like poly-L-lysine-polyethylene glycol (PLL-PEG) with averagedimensions of 12×150 nm is synthesized by slow addition of 9 volumes ofmRNA to 1 volume of CK₃₀-PEG_(10k) in acetate counterion buffer whilevortexing. (3) For synthesis of biodegradable gene carrier polymer,polyaspartic anhydride-co-ethylene glycol (PAE) is synthesized by ringopening polycondensation of N-(Benzyloxycarbonyl)-L-aspartic anhydrideand ethylene glycol. Then, the pendent amine of aspartic acid isdeprotected and protonated by acidification with hydrogen chloride andcondensed with mRNA. (4) For latest generation of nanoparticles, aliquotstock CK₃₀PEG_(10k) as ammonium acetate (1.25 mL; 6.4 mg/mL) is added tosiliconized Eppendorf tubes. Then mRNA is added slowly to CK₃₀PEG_(10k)(2.5 mg in 11.25 mL RNase-free H₂O) over 1-2 mins. After 15 mins, it isdiluted 1:2 in RNase-free H₂O.

Intratracheal Delivery.

Mice are anesthetized with 3% halothane (70% N₂O+30% O₂) in ananesthetic chamber and maintained with 1% halothane (70% N₂O+30% O₂)during operation using a nose cone. Trachea os exposed, and 50 μl ofmRNA complex is infused with 150 μl air into the lung through thetrachea using 250 μl Hamilton syringe (Hamilton, Reno, Nev.) with a 27 G½″ needle.

Results

To improve efficiency of delivery and expression of ψmRNA administeredvia the intratracheal (i.t.) route, ψmRNA is encapsulated innanoparticles. Nanoparticle packaging involves condensing andencapsulating DNA (for example) into particles that are smaller than thepore of the nuclear membrane, using chemicals including poly-L-lysineand polyethylene glycol. RNA is packaged into 4 different nanoparticleformulations (PEI, PLL, PAE, and CK₃₀PEG_(10k)), and efficiency of ψmRNAdelivery is compared for luciferase-encoding ψmRNA compare the(Luc-ψmRNA). Delivery kinetics and dose-response are then characterizedusing EPO-ψmRNA.

Example 22 Prevention of Restenosis by Delivery to the Carotid Artery ofRecombinant Heat Shock Protein-Encoding, Modified mRNA Materials andExperimental Methods

Experimental Design

RNA is administered to the carotid artery of rats by intra-arterialinjection near the time of balloon angioplasty, after which blood flowis reinstated. Rats are sacrificed 3 h following injection, carotidartery sections are excised, vascular endothelial cells are harvestedand homogenized, and luciferase activity is determined as described inabove Examples.

Results

Luciferase-encoding pseudouridine-modified RNA is administered to ratcarotid arteries. 3 hours later, luciferase RNA can be detected at thedelivery site but not the adjacent sites.

Next, this protocol is used to prevent restenosis of a blood vesselfollowing balloon angioplasty in an animal restenosis model, by deliveryof modified RNA encoding a heat shock protein, e.g. HSP70; a growthfactor (e.g. platelet-derived growth factor (PDGF), vascular endothelialgrowth factor (V-EGF), or insulin-like growth factor (IGF); or a proteinthat down-regulates or antagonizes growth factor signaling.Administration of modified RNA reduces incidence of restenosis.

Example 23 Treatment of Cystic Fibrosis by Delivery of CFTR-EncodingModified mRNA Molecules to Respiratory Epithelium

CFTR-encoding pseudouridine- or nucleoside-modified RNA is delivered, asdescribed in Example 13, to the lungs of a cystic fibrosis animal model,and its effect on the disease is assessed as described in Scholte B J,et al (Animal models of cystic fibrosis. J Cyst Fibros 2004; 3 Suppl 2:183-90) or Copreni E, et al, Lentivirus-mediated gene transfer to therespiratory epithelium: a promising approach to gene therapy of cysticfibrosis. Gene Ther 2004; 11 Suppl 1: 567-75). Administration of the RNAameliorates cystic fibrosis.

In additional experiments, modified mRNA molecules of the presentinvention are used to deliver to the lungs other recombinant proteins oftherapeutic value, e.g. via an inhaler that delivers RNA.

Example 24 Treatment of XLA by Delivery of ADA-Encoding Modified mRNAMolecules to Hematopoietic Cells

ADA-encoding pseudouridine- or nucleoside-modified RNA is delivered tothe hematopoietic cells of an X-linked agammaglobulinemia animal model,and its effect on the disease is assessed as described in Tanaka M,Gunawan F, et al, Inhibition of heart transplant injury and graftcoronary artery disease after prolonged organ ischemia by selectiveprotein kinase C regulators. J Thorac Cardiovasc Surg 2005; 129(5):1160-7) or Zonta S, Lovisetto F, et al, Uretero-neocystostomy in a swinemodel of kidney transplantation: a new technique. J Surg Res. 2005April; 124(2):250-5). Administration of the RNA is found to improve XLA.

Example 25 Prevention of Organ Rejection by Delivery ofImmuno-modulatory protein-encoding modified mRNA Molecules to aTransplant Site

Pseudouridine- or nucleoside-modified RNA encoding a cytokine, achemokine, or an interferon (e.g. IL-4, IL-13, IL-10, or TGF-β) isdelivered to the transplant site of an organ transplant rejection animalmodel, and its effect on the incidence of rejection is assessed asdescribed in Yu P W, Tabuchi R S et al, Sustained correction of B-celldevelopment and function in a murine model of X-linkedagammaglobulinemia (XLA) using retroviral-mediated gene transfer. Blood.2004 104(5): 1281-90) or Satoh M, Mizutani A et al, X-linkedimmunodeficient mice spontaneously produce lupus-related anti-RNAhelicase A autoantibodies, but are resistant to pristane-induced lupus.Int Immunol 2003, 15(9):1117-24). Administration of the RNA reducesincidence of transplant rejection.

Example 26 Treatment of Niemann-Pick Disease, Mucopolysaccharidosis, andOther Inborn Metabolic Errors by Delivery of Modified mRNA to BodyTissues

Sphingomyelinase-encoding pseudouridine- or nucleoside-modified RNA isdelivered to the lung, brain, or other tissue of Niemann-Pick diseaseType A and B animal models, and its effect on the disease is assessed asdescribed in Passini M A, Macauley S L, et al, AAV vector-mediatedcorrection of brain pathology in a mouse model of Niemann-Pick Adisease. Mol Ther 2005; 11(5): 754-62) or Buccoliero R, Ginzburg L, etal, Elevation of lung surfactant phosphatidylcholine in mouse models ofSandhoff and of Niemann-Pick A disease. J Inherit Metab Dis 2004; 27(5):641-8). Administration of the RNA is found to improve the disease.

Pseudouridine- or nucleoside-modified RNA encoding alpha-L-iduronidase,iduronate-2-sulfatase, or a related enzyme is delivered to the bodytissues of a mucopolysaccharidosis animal model of, and its effect onthe disease is assessed as described in Simonaro C M, D'Angelo M, et al,Joint and bone disease in mucopolysaccharidoses VI and VII:identification of new therapeutic targets and biomarkers using animalmodels. Pediatr Res 2005; 57(5 Pt 1): 701-7) or McGlynn R, Dobrenis K,et al, Differential subcellular localization of cholesterol,gangliosides, and glycosaminoglycans in murine models ofmucopolysaccharide storage disorders. J Comp Neurol 2004 20; 480(4):415-26). Administration of the RNA ameliorates the disease.

In additional experiments, modified mRNA molecules of the presentinvention are used to provide clotting factors (e.g. for hemophiliacs).

In additional experiments, modified mRNA molecules of the presentinvention are used to provide acid-b-glucosidase for treating Gaucher's.

In additional experiments, modified mRNA molecules of the presentinvention are used to provide alpha-galactosidase A for treating Fabry'sdiseases.

In additional experiments, modified mRNA molecules of the presentinvention are used to provide cytokines for treatment of infectiousdiseases.

In additional experiments, modified mRNA molecules of the presentinvention are used to correct other inborn errors of metabolism, byadministration of mRNA molecules encoding, e.g. ABCA4; ABCD3; ACADM;AGL; AGT; ALDH4A1; ALPL; AMPD1; APOA2; AVSD1; BRCD2; C1QA; C1QB; C1QG;CBA; C8B; CACNA1S; CCV; CD3Z; CDC2L1; CHML; CHS1; CIAS1; CLCNKB; CMD1A;CMH2; CMM; COL11A1; COL8A2; COL9A2; CPT2; CRB1; CSE; CSF3R; CTPA; CTSK;DBT; DIOL; DISC1; DPYD; EKV; ENO1; ENO1P; EPB41; EPHX1; F13B; F5;FCGR2A; FCGR2B; FCGR3A; FCHL; FH; FMO3; FMO4; FUCA1; FY; GALE; GBA;GFND; GJA8; GJB3; GLC3B; HF1; HMGCL; HPC1; HRD; HRPT2; HSD3B2; HSPG2;KCNQ4; KCS; KIF1B; LAMB3; LAMC2; LGMD1B; LMNA; LOR; MCKD1; MCL1; MPZ;MTHFR; MTR; MUTYH; MYOC; NB; NCF2; NEM1; NPHS2; NPPA; NRAS; NTRK1;OPTA2; PBX1; PCHC; PGD; PHA2A; PHGDH; PKLR; PKP1; PLA2G2A; PLOD; PPDX;PPT1; PRCC; PRG4; PSEN2; PTOS1; REN; RFX5; RHD; RMD1; RPE65; SCCD;SERPINC1; SJS1; SLC19A2; SLC2A1; SPG23; SPTA1; TAL1; TNFSF6; TNNT2;TPM3; TSHB; UMPK; UOX; UROD; USH2A; VMGLOM; VWS; WS2B; ABCB11; ABCG5;ABCG8; ACADL; ACP1; AGXT; AHHR; ALMS1; ALPP; ALS2; APOB; BDE; BDMR; BJS;BMPR2; CHRNA1; CMCWTD; CNGA3; COL3A1; COL4A3; COL4A4; COL6A3; CPS1;CRYGA; CRYGEP1; CYP1B1; CYP27A1; DBI; DES; DYSF; EDAR; EFEMP1; EIF2AK3;ERCC3; FSHR; GINGF; GLC1B; GPD2; GYPC; HADHA; HADHB; HOXD13; HPE2; IGKC;IHH; IRS1; ITGA6; KHK; KYNU; LCT; LHCGR; LSFC; MSH2; MSH6; NEB; NMTC;NPHP1; PAFAH1P1; PAX3; PAX8; PMS1; PNKD; PPH1; PROC; REG1A; SAG; SFTPB;SLC11A1; SLC3A1; SOS1; SPG4; SRD5A2; TCL4; TGFA; TMD; TPO; UGT1A@; UV24;WSS; XDH; ZAP70; ZFHX1B; ACAA1; AGS1; AGTR1; AHSG; AMT; ARMET; BBS3;BCHE; BCPM; BTD; CASR; CCR2; CCR5; CDL1; CMT2B; COL7A1; CP; CPO; CRV;CTNNB1; DEM; ETM1; FANCD2; FIH; FOXL2; GBE1; GLB1; GLC1C; GNAI2; GNAT1;GP9; GPX1; HGD; HRG; ITIH1; KNG; LPP; LRS1; MCCC1; MDS1; MHS4; MITF;MLH1; MYL3; MYMY; OPA1; P2RY12; PBXP1; PCCB; POU1F1; PPARG; PRO51;PTHR1; RCA1; RHO; SCAT; SCLC1; SCN5A; SI; SLC25A20; SLC2A2; TF; TGFBR2;THPO; THRB; TKT; TM4SF1; TRH; UMPS; UQCRC1; USH3A; VHL; WS2A; XPC;ZNF35; ADH1B; ADH1C; AFP; AGA; AIH2; ALB; ASMD; BFHD; CNGA1; CRBM; DCK;DSPP; DTDP2; ELONG; ENAM; ETFDH; EVC; F11; FABP2; FGA; FGB; FGFR3; FGG;FSHMD1A; GC; GNPTA; GNRHR; GYPA; HCA; HCL2; HD; HTN3; HVBS6; IDUA; IF;JPD; KIT; KLKB1; LQT4; MANBA; MLLT2; MSX1; MTP; NR3C2; PBT; PDE6B; PEE1;PITX2; PKD2; QDPR; SGCB; SLC25A4; SNCA; SOD3; STATH; TAPVR1; TYS; WBS2;WFS1; WHCR; ADAMTS2; ADRB2; AMCN; AP3B1; APC; ARSB; B4GALT7; BHR1; C6;C7; CCAL2; CKN1; CMDJ; CRHBP; CSF1R; DHFR; DIAPH1; DTR; EOS; EPD; ERVR;F12; FBN2; GDNF; GHR; GLRA1; GM2A; HEXB; HSD17B4; ITGA2; KFS; LGMD1A;LOX; LTC4S; MAN2A1; MCC; MCCC2; MSH3; MSX2; NR3C1; PCSK1; PDE6A; PFBI;RASA1; SCZD1; SDHA; SGCD; SLC22A5; SLC26A2; SLC6A3; SM1; SMA@; SMN1;SMN2; SPINK5; TCOF1; TELAB1; TGFBI; ALDH5A1; ARG1; AS; ASSP2; BCKDHB;BF; C2; C4A; CDKN1A; COL10A1; COL11A2; CYP21A2; DYX2; EJM1; ELOVL4;EPM2A; ESR1; EYA4; F13A1; FANCE; GCLC; GJA1; GLYS1; GMPR; GSE; HCR; HFE;HLA-A; HLA-DPB1; HLA-DRA; HPFH; ICS1; IDDM1; IFNGR1; IGAD1; IGF2R; ISCW;LAMA2; LAP; LCA5; LPA; MCDR1; MOCS1; MUT; MYB; NEU1; NKS1; NYS2; OA3;ODDD; OFC1; PARK2; PBCA; PBCRA1; PDB1; PEX3; PEX6; PEX7; PKHD1; PLA2G7;PLG; POLH; PPAC; PSORS1; PUJO; RCD1; RDS; RHAG; RP14; RUNX2; RWS; SCA1;SCZD3; SIASD; SOD2; ST8; TAP1; TAP2; TFAP2B; TNDM; TNF; TPBG; TPMT;TULP1; WISP3; AASS; ABCB1; ABCB4; ACHE; AQP1; ASL; ASNS; AUTS1; BPGM;BRAF; C7orf2; CACNA2D1; CCM1; CD36; CFTR; CHORDOMA; CLCN1; CMH6; CMT2D;COL1A2; CRS; CYMD; DFNA5; DLD; DYT11; EEC1; ELN; ETV1; FKBP6; GCK;GHRHR; GHS; GLI3; GPDS1; GUSB; HLXB9; HOXA13; HPFH2; HRX; IAB; IMMP2L;KCNH2; LAMB1; LEP; MET; NCF1; NM; OGDH; OPN1SW; PEX1; PGAM2; PMS2; PON1;PPP1R3A; PRSS1; PTC; PTPN12; RP10; RP9; SERPINE1; SGCE; SHFM1; SHH;SLC26A3; SLC26A4; SLOS; SMAD1; TBXAS1; TWIST; ZWS1; ACHM3; ADRB3; ANK1;CA1; CA2; CCAL1; CLN8; CMT4A; CNGB3; COH1; CPP; CRH; CYP11B1; CYP11B2;DECR1; DPYS; DURST; EBS1; ECA1; EGI; EXT1; EYA1; FGFR1; GNRH1; GSR;GULOP; HR; KCNQ3; KFM; KWE; LGCR; LPL; MCPH1; MOS; MYC; NAT1; NAT2;NBS1; PLAT; PLEC1; PRKDC; PXMP3; RP1; SCZD6; SFTPC; SGM1; SPG5A; STAR;TG; TRPS1; TTPA; VMD1; WRN; ABCA1; ABL1; ABO; ADAMTS13; AK1; ALAD;ALDH1A1; ALDOB; AMBP; AMCD1; ASS; BDMF; BSCL; C5; CDKN2A; CHAC; CLA1;CMD1B; COL5A1; CRAT; DBH; DNAI1; DYS; DYT1; ENG; FANCC; FBP1; FCMD;FRDA; GALT; GLDC; GNE; GSM1; GSN; HSD17B3; HSN1; IBM2; INVS; JBTS1;LALL; LCCS1; LCCS; LGMD2H; LMX1B; MLLT3; MROS; MSSE; NOTCH1; ORM1;PAPPA; PIP5K1B; PTCH; PTGS1; RLN1; RLN2; RMRP; ROR2; RPD1; SARDH;SPTLC1; STOM; TDFA; TEK; TMC1; TRIM32; TSC1; TYRP1; XPA; CACNB2;COL17A1; CUBN; CXCL12; CYP17; CYP2C19; CYP2C9; EGR2; EMX2; ERCC6; FGFR2;HK1; HPS1; IL2RA; LGI1; L1PA; MAT1A; MBL2; MKI67; MXI1; NODAL; OAT;OATL3; PAX2; PCBD; PEO1; PHYH; PNLIP; PSAP; PTEN; RBP4; RDPA; RET;SFTPA1; SFTPD; SHFM3; SIAL; THC2; TLX1; TNFRSF6; UFS; UROS; AA; ABCC8;ACAT1; ALX4; AMPD3; ANC; APOA1; APOA4; APOC3; ATM; BSCL2; BWS; CALCA;CAT; CCND1; CD3E; CD3G; CD59; CDKN1C; CLN2; CNTF; CPT1A; CTSC; DDB1;DDB2; DHCR7; DLAT; DRD4; ECB2; ED4; EVR1; EXT2; F2; FSHB; FTH1; G6PT1;G6PT2; GIF; HBB; HBBP1; HBD; HBE1; HBG1; HBG2; HMBS; HND; HOMG2; HRAS;HVBS1; IDDM2; IGER; INS; JBS; KCNJ11; KCNJ1; KCNQ1; LDHA; LRP5; MEN1;MLL; MYBPC3; MYO7A; NNO1; OPPG; OPTB1; PAX6; PC; PDX1; PGL2; PGR; PORC;PTH; PTS; PVRL1; PYGM; RAG1; RAG2; ROM1; RRAS2; SAA1; SCA5; SCZD2; SDHD;SERPING1; SMPD1; TCIRG1; TCL2; TECTA; TH; TREH; TSG101; TYR; USH1C;VMD2; VRNI; WT1; WT2; ZNF145; A2M; AAAS; ACADS; ACLS; ACVRL1; ALDH2;AMHR2; AOM; AQP2; ATD; ATP2A2; BDC; C1R; CD4; CDK4; CNA1; COL2A1;CYP27B1; DRPLA; ENUR2; FEOM1; FGF23; FPF; GNB3; GNS; HAL; HBP1; HMGA2;HMN2; HPD; IGF1; KCNA1; KERA; KRAS2; KRT1; KRT2A; KRT3; KRT4; KRT5;KRT6A; KRT6B; KRTHB6; LDHB; LYZ; MGCT; MPE; MVK; MYL2; OAP; PAH; PPKB;PRB3; PTPN11; PXR1; RLS; RSN; SAS; SAX1; SCA2; SCNN1A; SMAL; SPPM;SPSMA; TBX3; TBX5; TCF1; TPI1; TSC3; ULR; VDR; VWF; ATP7B; BRCA2; BRCD1;CLN5; CPB2; ED2; EDNRB; ENUR1; ERCC5; F10; F7; GJB2; GJB6; IPF1; MBS1;MCOR; NYS4; PCCA; RB1; RHOK; SCZD7; SGCG; SLC10A2; SLC25A15; STARP1;ZNF198; ACHM1; ARVD1; BCH; CTAA1; DAD1; DFNB5; EML1; GALC; GCH1; IBGC1;IGH@; IGHC group; IGHG1; IGHM; IGHR; IV; LTBP2; MCOP; MJD; MNG1; MPD1;MPS3C; MYH6; MYH7; NP; NPC2; PABPN1; PSEN1; PYGL; RPGRIP1; SERPINA1;SERPINA3; SERPINA6; SLC7A7; SPG3A; SPTB; TCL1A; TGM1; TITF1; TMIP; TRA@;TSHR; USH1A; VP; ACCPN; AHO2; ANCR; B2M; BBS4; BLM; CAPN3; CDAN1; CDAN3;CLN6; CMH3; CYP19; CYP1A1; CYP1A2; DYX1; EPB42; ETFA; EYCL3; FAH; FBN1;FES; HCVS; HEXA; IVD; LCS1; LIPC; MYO5A; OCA2; OTSC1; PWCR; RLBP1;SLC12A1; SPG6; TPM1; UBE3A; WMS; ABCC6; ALDOA; APRT; ATP2A1; BBS2;CARD15; CATM; CDH1; CETP; CHST6; CLN3; CREBBP; CTH; CTM; CYBA; CYLD;DHS; DNASE1; DPEP1; ERCC4; FANCA; GALNS; GAN; HAGH; HBA1; HBA2; HBHR;HBQ1; HBZ; HBZP; HP; HSD11B2; IL4R; LIPB; MC1R; MEFV; MHC2TA; MLYCD;MMVP1; PHKB; PHKG2; PKD1; PKDTS; PMM2; PXE; SALL1; SCA4; SCNN1B; SCNN1G;SLC12A3; TAT; TSC2; VDI; WT3; ABR; ACACA; ACADVL; ACE; ALDH3A2; APOH;ASPA; AXIN2; BCL5; BHD; BLMH; BRCA1; CACD; CCA1; CCZS; CHRNB1; CHRNE;CMT1A; COL1A1; CORDS; CTNS; EPX; ERBB2; G6PC; GAA; GALK1; GCGR; GFAP;GH1; GH2; GP1BA; GPSC; GUCY2D; ITGA2B; ITGB3; ITGB4; KRT10; KRT12;KRT13; KRT14; KRT14L1; KRT14L2; KRT14L3; KRT16; KRT16L1; KRT16L2; KRT17;KRT9; MAPT; MDB; MDCR; MGI; MHS2; MKS1; MPO; MYO15A; NAGLU; NAPB; NF1;NME1; P4HB; PAFAH1B1; PECAM1; PEX12; PHB; PMP22; PRKAR1A; PRKCA;PRKWNK4; PRP8; PRPF8; PTLAH; RARA; RCV1; RMSA1; RP17; RSS; SCN4A;SERPINF2; SGCA; SGSH; SHBG; SLC2A4; SLC4A1; SLC6A4; SMCR; SOST; SOX9;SSTR2; SYM1; SYNS1; TCF2; THRA; TIMP2; TOC; TOP2A; TP53; TRIM37; VBCH;ATP8B1; BCL2; CNSN; CORD1; CYB5; DCC; F5F8D; FECH; FEO; LAMA3; LCFS2;MADH4; MAFD1; MC2R; MCL; MYP2; NPC1; SPPK; TGFBRE; TGIF; TTR; AD2; AMH;APOC2; APOE; ATHS; BAX; BCKDHA; BCL3; BFIC; C3; CACNA1A; CCO; CEACAM5;COMP; CRX; DBA; DDU; DFNA4; DLL3; DM1; DMWD; E11S; ELA2; EPOR; ERCC2;ETFB; EXT3; EYCL1; FTL; FUT1; FUT2; FUT6; GAMT; GCDH; GPI; GUSM; HB1;HCL1; HHC2; HHC3; ICAM3; INSR; JAK3; KLK3; LDLR; LHB; LIG1; LOH19CR1;LYL1; MAN2B1; MCOLN1; MDRV; MLLT1; NOTCH3; NPHS1; OFC3; OPA3; PEPD;PRPF31; PRTN3; PRX; PSG1; PVR; RYR1; SLC5A5; SLC7A9; STK11; TBXA2R;TGFB1; TNNI3; TYROBP; ADA; AHCY; AVP; CDAN2; CDPD1; CHED1; CHED2;CHRNA4; CST3; EDN3; EEGV1; FTLL1; GDF5; GNAS; GSS; HNF4A; JAG1; KCNQ2;MKKS; NBIA1; PCK1; PI3; PPCD; PPGB; PRNP; THBD; TOP1; AIRE; APP; CBS;COL6A1; COL6A2; CSTB; DCR; DSCR1; FPDMM; HLCS; HPE1; ITGB2; KCNE1; KNO;PRSS7; RUNX1; SOD1; TAM; ADSL; ARSA; BCR; CECR; CHEK2; COMT; CRYBB2;CSF2RB; CTHM; CYP2D6; CYP2D7P1; DGCR; DIA1; EWSR1; GGT1; MGCR; MN1;NAGA; NF2; OGS2; PDGFB; PPARA; PRODH; SCO2; SCZD4; SERPIND1; SLC5A1;SOX10; TCN2; TIMP3; TST; VCF; ABCD1; ACTL1; ADFN; AGMX2; AHDS; AIC;AIED; AIH3; ALAS2; AMCD; AMELX; ANOP1; AR; ARAF1; ARSC2; ARSE; ARTS;ARX; ASAT; ASSP5; ATP7A; ATRX; AVPR2; BFLS; BGN; BTK; BZX; C1HR;CACNA1F; CALB3; CBBM; CCT; CDR1; CFNS; CGF1; CHM; CHR39c; CIDX; CLA2;CLCN5; CLS; CMTX2; CMTX3; CND; COD1; COD2; COL4A5; COL4A6; CPX; CVD1;CYBB; DCX; DFN2; DFN4; DFN6; DHOF; DIAPH2; DKC1; DMD; DSS; DYT3; EBM;EBP; ED1; ELK1; EMD; EVR2; F8; F9; FCP1; FDPSL5; FGD1; FGS1; FMR1; FMR2;G6PD; GABRA3; GATA1; GDI1; GDXY; GJB1; GK; GLA; GPC3; GRPR; GTD; GUST;HMS1; HPRT1; HPT; HTC2; HTR2c; HYR; IDS; IHG1; IL2RG; INDX; IP1; IP2;JMS; KAL1; KFSD; L1CAM; LAMP2; MAA; MAFD2; MAOA; MAOB; MCF2; MCS; MEAX;MECP2; MF4; MGC1; MIC5; MID1; MLLT7; MLS; MRSD; MRX14; MRX1; MRX20;MRX2; MRX3; MRX40; MRXA; MSD; MTM1; MYCL2; MYP1; NDP; NHS; NPHL1; NROB1;NSX; NYS1; NYX; OA1; OASD; OCRL; ODT1; OFD1; OPA2; OPD1; OPEM; OPN1LW;OPN1MW; OTC; P3; PDHA1; PDR; PFC; PFKFB1; PGK1; PGK1P1; PGS; PHEX;PHKA1; PHKA2; PHP; PIGA; PLP1; POF1; POLA; POU3F4; PPMX; PRD; PRPS1;PRPS2; PRS; RCCP2; RENBP; RENS1; RP2; RP6; RPGR; RPS4X; RPS6KA3; RS1;S11; SDYS; SEDL; SERPINA7; SH2D1A; SHFM2; SLC25A5; SMAX2; SRPX; SRS;STS; SYN1; SYP; TAF1; TAZ; TBX22; TDD; TFE3; THAS; THC; TIMM8A; TIMP1;TKCR; TNFSF5; UBE1; UBE2A; WAS; WSN; WTS; WWS; XIC; XIST; XK; XM; XS;ZFX; ZIC3; ZNF261; ZNF41; ZNF6; AMELY; ASSP6; AZF1; AZF2; DAZ; GCY;RPS4Y; SMCY; SRY; ZFY; ABAT; AEZ; AFA; AFD1; ASAH1; ASD1; ASMT; CCAT;CECR9; CEPA; CLA3; CLN4; CSF2RA; CTS1; DF; DIH1; DWS; DYT2; DYT4; EBR3;ECT; EEF1A1L14; EYCL2; FANCB; GCSH; GCSL; GIP; GTS; HHG; HMI; HOAC;HOKPP2; HRPT1; HSD3B3; HTC1; HV1S; ICHQ; ICR1; ICR5; IL3RA; KAL2; KMS;KRT18; KSS; LCAT; LHON; LIMM; MANBB; MCPH2; MEB; MELAS; MIC2; MPFD; MS;MSS; MTATP6; MTCO1; MTCO3; MTCYB; MTND1; MTND2; MTND4; MTND5; MTND6;MTRNR1; MTRNR2; MTTE; MTTG; MTTI; MTTK; MTTL1; MTTL2; MTTN; MTTP; MTTS1;NAMSD; OCD1; OPD2; PCK2; PCLD; PCOS1; PFKM; PKD3; PRCA1; PRO1; PROP1;RBS; RFXAP; RP; SHOX; SLC25A6; SPG5B; STO; SUOX; THM; or TTD.

Example 27 Treatment Of Vasospasm by Delivery of iNOS-Encoding ModifiedmRNA Molecules to Body Tissues

Inducible nitric oxide synthase (iNOS)-encoding pseudouridine- ornucleoside-modified RNA is delivered to the vascular endothelium ofvasospasm animals models (e.g. subarachnoid hemorrhage), and its effecton the disease is assessed as described in Pradilla G, Wang P P, et al,Prevention of vasospasm by anti-CD11/CD18 monoclonal antibody therapyfollowing subarachnoid hemorrhage in rabbits. J Neurosurg 2004; 101(1):88-92) or Park S, Yamaguchi M, et al, Neurovascular protection reducesearly brain injury after subarachnoid hemorrhage. Stroke 2004; 35(10):2412-7). Administration of the RNA ameliorates the disease.

Example 28 Restoration of Hair Growth by Delivery of Modified mRNAEncoding an Immunosuppressive Protein

Pseudouridine- or nucleoside-modified RNA encoding a telomerase or animmunosuppressive protein (e.g. α-MSH, TGF-β1, or IGF-I is delivered tohair follicles of animals used as models of hair loss or balding, andits effect on hair growth is assessed as described in Jiang J, Tsuboi R,et al, Topical application of ketoconazole stimulates hair growth inC3H/HeN mice. J Dermatol 2005; 32(4): 243-7) or McElwee K J,Freyschmidt-Paul P, et al, Transfer of CD8(+) cells induces localizedhair loss whereas CD4(+)/CD25(−) cells promote systemic alopecia greataand CD4(+)/CD25(+) cells blockade disease onset in the C3H/HeJ mousemodel. J Invest Dermatol 2005; 124(5): 947-57). Administration of theRNA restores hair growth.

Example 29 Synthesis of an In Vitro-Transcribed RNA Molecule withAltered Nucleosides Containing an siRNA

A double-stranded RNA (dsRNA) molecule comprising pseudouridine or amodified nucleoside and further comprising a small interfering RNA(siRNA) or short hairpin RNA (shRNA) is synthesized by the followingprocedure: Complementary RNA strands with the desired sequencecontaining uridine or 1 or more modified nucleosides are synthesized byin vitro transcription (e.g. by T7, SP6, or T3 phage RNA polymerase) asdescribed in Example 2. dsRNA molecules exhibit reduced immunogenicity.In other experiments, the dsRNA molecules are designed to be processedby a cellular enzyme to yield the desired siRNA or shRNA. Because dsRNAmolecules of several hundred nucleotides are easily synthesized, eachdsRNA may also be designed to contain several siRNA or shRNA molecules,to facilitate delivery of multiple siRNA or shRNA to a single targetcell.

Example 30 Use of an In Vitro-Transcribed RNA Molecule with AlteredNucleosides to Deliver siRNA

The dsRNA molecule of the previous Example is complexed with atransfection reagent (e.g a cationic transfection reagent, a lipid-basedtransfection reagent, a protein-based transfection reagent, apolyethyleneimine based transfection reagent, or calcium phosphate) anddelivered to a target cell of interest. Enzymes in or on the surface ofthe target cell degrade the dsRNA to the desired siRNA or shRNAmolecule(s). This method effectively silences transcription of 1 or morecellular genes corresponding to the siRNA or shRNA sequence(s).

Example 31 Testing the Effect of Additional Nucleoside Modifications onRNA Immunogenicity and Efficiency of Translation

Additional nucleoside modifications are introduced into invitro-transcribed RNA, using the methods described above in Examples 2and 7, and their effects on immunogenicity translation efficiency aretested as described in Examples 1-8 and 9-15, respectively. Certainadditional modifications are found to decrease immunogenicity andenhance translation. These modifications are additional embodiments ofmethods and compositions of the present invention.

Modifications tested include, e.g.:

m¹A; m²A; Am; ms²m⁶A; i⁶A; ms²i6A; io⁶A; ms²io⁶A; g⁶A; t⁶A; ms²t⁶A;m⁶t⁶A; hn⁶A; ms²hn⁶A; Ar(p); I; m¹I; m¹Im; m³C; Cm; s²C; act; f⁵C; m⁵Cm;ac⁴Cm; k²C; m¹G; m²G; m⁷G; Gm; m² ₂G; m²Gm; m² ₂Gm; Gr(p); yW; o₂yW;OHyW; OHyW*; imG; mimG; Q; oQ; galQ; manQ; preQ₀; preQ₁; G⁺; D; m⁵Um;m¹Ψ; Ψm; s⁴U; m⁵s²U; s²Um; acp³U; ho⁵U; mo⁵U; cmo⁵U; mcmo⁵U; chm⁵U;mchm⁵U; mcm⁵U; mcm⁵Um; mcm⁵s²U; nm⁵s²U; mnm⁵U; mnm⁵s²U; mnm⁵se²U; ncm⁵U;ncm⁵Um; cmnm⁵U; cmnm⁵Um; cmnm⁵s²U; m⁶ ₂A; Im; m⁴C; m⁴Cm; hm⁵C; m³U;m¹acp³Ψ; cm⁵U; m⁶Am; m⁶ ₂Am; m^(2,7)G; m^(2,2,7)G; M³UM; M⁵D; M³Ψ; f⁵Cm;M¹Gm; M¹Am; τm⁵U; τm⁵s²U; imG-14; imG2; and ac⁶A.

What is claimed is:
 1. A composition comprising an in vitro-synthesizedmodified RNA comprising an open reading frame that encodes a protein ofinterest for translation in a mammalian cell, wherein said invitro-synthesized modified RNA comprises a modified nucleoside selectedfrom the group consisting of (i) 1-methypseudouridine (m¹Ψ) and (ii)pseudouridine (Ψ).
 2. The composition of claim 1, wherein said invitro-synthesized modified RNA further comprises a 5′-terminal capcomprising N⁷-methylguanine and a poly-A tail.
 3. The composition ofclaim 2, wherein the cap in the in vitro-synthesized modified RNAcomprises m⁷GpppG cap or 3′-O-methyl-m⁷GpppG cap.
 4. The composition ofclaim 1, wherein said in vitro-synthesized modified RNA furthercomprises a cap-independent translational enhancer.
 5. The compositionof claim 1, wherein said in vitro-synthesized modified RNA furthercomprises 5′ and 3′ untranslated regions (UTRs) that enhancetranslation.
 6. The composition of claim 5, wherein said 5′ and 3′ UTRscomprise at least one UTR selected from the group consisting of abeta-globin 5′ UTR, a tobacco etch virus (TEV) 5′ UTR, and a beta-globin3′ UTR.
 7. The composition of claim 1, wherein said composition ispresent in a mammalian cell that is in culture, in a tissue, or in vivoin a mammal.
 8. The composition of claim 7, wherein the mammalian cellis a cell selected from the group consisting of an antigen-presentingcell, a dendritic cell, a macrophage, a neural cell, a brain cell, anastrocyte, a microglial cell, and a neuron, a spleen cell, a lymphoidcell, a lung cell, a lung epithelial cell, a skin cell, a keratinocyte,an endothelial cell, an alveolar cell, an alveolar macrophage, analveolar pneumocyte, a vascular endothelial cell, a mesenchymal cell, anepithelial cell, a colonic epithelial cell, a hematopoietic cell, a bonemarrow cell, a Claudius cell, Hensen cell, Merkel cell, Muller cell,Paneth cell, Purkinje cell, Schwann cell, Sertoli cell, acidophil cell,acinar cell, adipoblast, adipocyte, brown or white alpha cell, amacrinecell, beta cell, capsular cell, cementocyte, chief cell, chondroblast,chondrocyte, chromaffin cell, chromophobic cell, corticotroph, deltacell, Langerhans cell, follicular dendritic cell, enterochromaffin cell,ependymocyte, epithelial cell, basal cell, squamous cell, endothelialcell, transitional cell, erythroblast, erythrocyte, fibroblast,fibrocyte, follicular cell, germ cell, gamete, ovum, spermatozoon,oocyte, primary oocyte, secondary oocyte, spermatid, spermatocyte,primary spermatocyte, secondary spermatocyte, germinal epithelium, giantcell, glial cell, astroblast, astrocyte, oligodendroblast,oligodendrocyte, glioblast, goblet cell, gonadotroph, granulosa cell,haemocytoblast, hair cell, hepatoblast, hepatocyte, hyalocyte,interstitial cell, juxtaglomerular cell, keratinocyte, keratocyte,lemmal cell, leukocyte, granulocyte, basophil, eosinophil, neutrophil,lymphoblast, B-lymphoblast, T-lymphoblast, lymphocyte, B-lymphocyte,T-lymphocyte, helper induced T-lymphocyte, Th1 T-lymphocyte, Th2T-lymphocyte, natural killer cell, thymocyte, macrophage, Kupffer cell,alveolar macrophage, foam cell, histiocyte, luteal cell, lymphocyticstem cell, lymphoid cell, lymphoid stem cell, macroglial cell,mammotroph, mast cell, medulloblast, megakaryoblast, megakaryocyte,melanoblast, melanocyte, mesangial cell, mesothelial cell,metamyelocyte, monoblast, monocyte, mucous neck cell, myoblast, myocyte,muscle cell, cardiac muscle cell, skeletal muscle cell, smooth musclecell, myelocyte, myeloid cell, myeloid stem cell, myoblast,myoepithelial cell, myofibrobast, neuroblast, neuroepithelial cell,neuron, odontoblast, osteoblast, osteoclast, osteocyte, oxyntic cell,parafollicular cell, paraluteal cell, peptic cell, pericyte, peripheralblood mononuclear cell, phaeochromocyte, phalangeal cell, pinealocyte,pituicyte, plasma cell, platelet, podocyte, proerythroblast,promonocyte, promyeloblast, promyelocyte, pronormoblast, reticulocyte,retinal pigment epithelial cell, retinoblast, small cell, somatotroph,stein cell, sustentacular cell, teloglial cell, and a zymogenic cell. 9.The composition of claim 1, wherein said in vitro-synthesized modifiedRNA induces a detectably lower innate immune response than the samequantity of in vitro-synthesized unmodified RNA that exhibits the samesequence except with uridine in place of said modified nucleosideselected from the group consisting of (i) 1-methypseudouridine (m¹Ψ) and(ii) pseudouridine (Ψ).
 10. The composition of claim 9, wherein saiddetectably lower innate immune response is detected by a method selectedfrom the group consisting of: (i) detecting that repeatedly contactingthe mammalian cell with an amount of the modified RNA that results indetectable expression of the encoded protein after a single contactingdoes not detectably reduce expression of the protein, whereas repeatedlycontacting the mammalian cell with the same quantity of the unmodifiedRNA does detectably reduce expression of the encoded protein; (ii)detecting that the modified RNA results in a lower level ofself-phosphorylation of RNA-activated protein kinase (PKR) and/orphosphorylation of the eukaryotic translation initiation factor (eIF2α)compared to the same quantity of the unmodified RNA counterpart based onin vitro phosphorylation assays; (iii) detecting that the quantity ofone or more cytokines induced by the mammalian cell in response tounmodified RNA is higher than the quantity of said one or more cytokinesinduced by the mammalian cell in response to said modified RNAcounterpart; (iv) detecting a difference in the level of expression ofone or more dendritic cell (DC) activation markers in response to theunmodified RNA compared to the level of expression of said one or moreDC activation markers in response to the same quantity of said modifiedRNA; (v) detecting a higher relative ability of said modified RNA to actas an adjuvant for an adaptive immune response compared to the samequantity of unmodified RNA counterpart; (vi) detecting a higher level ofactivation of toll-like receptor (TLR) signaling molecules in responseto unmodified RNA compared to the same quantity of said modified RNA;and/or (vii) determining the quantity of the modified RNA to elicit animmune response measured in any of cells (i)-(vi) compared to thequantity of unmodified RNA to elicit the same immune response.
 11. Thecomposition of claim 10, wherein: said one or more cytokines in (iii)are selected from the group consisting of IL-12, IFN-.alpha.,TNF-.alpha., RANTES, MIP-1.alpha., MIP-1.beta., IL-6, IFN-.beta., andIL-8; said DC activation markers in (iv) are selected from the groupconsisting of: CD83, HLA-DR, CD80, and CD86; or said TLR signalingmolecules in (vi) are selected from the group consisting of: TLR3, TLR7,and TLR8 signaling molecules.
 12. The composition of claim 10, whereinsaid detectably lower innate immune response induced by said modifiedRNA is at least 2-fold lower than the innate immune response induced bysaid unmodified RNA using at least one of said cells for determining ormeasuring said detectable decrease in immunogenicity.
 13. Thecomposition of claim 1, wherein said in vitro-synthesized modified RNAexhibits enhanced ability to produce said encoded protein of interest insaid mammalian cell compared to the same quantity of an invitro-synthesized unmodified RNA that exhibits the same sequence butwith uridine in place of said pseudouridine modified nucleoside, whereinsaid enhanced ability to produce said protein of interest is determinedby measuring a higher level of either the amount of protein or theamount of enzymatic activity or other biological effect produced at oneor more times after contacting a mammalian with said modified RNAcompared to the corresponding amount of protein or amount of enzymaticactivity or other biological effect produced in the same or equivalentmammalian cell at the same times after contacting with the same quantityof the unmodified RNA.
 14. The composition of claim 13, wherein abilityto produce said encoded protein of interest in said mammalian cell isenhanced by at least 2-fold for said modified RNA compared to saidunmodified RNA.
 15. The composition of claim 1, wherein said invitro-synthesized modified RNA is encapsulated in a nanoparticle,polymer, lipid, cholesterol, or a cell penetrating peptide.
 16. Thecomposition of claim 1, wherein said in vitro-synthesized modified RNAencodes a protein selected from the group consisting of erythropoietin(EPO), a detectable enzyme selected from firefly luciferase, Renillaluciferase, bacterial beta-galactosidase (lacZ), green fluorescentprotein (GFP), MYC, SRY, MCOP, platelet-derived growth factor (PDGF),vascular endothelial growth factor (VEGF), transforming growthfactor-beta1 (TGF-beta1), insulin-like growth factor (IGF),alpha-melanocyte-stimulating hormone (alpha-MSH), insulin-like growthfactor-I (IGF-I), IL-4, IL-13, IL-10, inducible nitric oxide synthase(iNOS), a heat shock protein, cystic fibrosis transmembrane conductanceregulator (CFTR), an enzyme with antioxidant activity, catalase,phospholipid hydroperoxide glutathione peroxidase, superoxidedismutase-1, superoxide dismutase-2, Bruton's tyrosine kinase, adenosinedeaminase, ecto-nucleoside triphosphate diphosphydrolase, ABCA4, ABCD3,ACADM, AGL, AGT, ALDH4A1, ALPL, AMPD1, APOA2, AVSD1, BRCD2, C1QA, C1QB,C1QG, C8A, C8B, CACNA1S, CCV, CD3Z, CDC2L1, CHML, CHS1, CIAS1, CLCNKB,CMD1A, CMH2, CMM, COL11A1, COL8A2, COL9A2, CPT2, CRB1, CSE, CSF3R, CTPA,CTSK, DBT, DIOL DISCI, DPYD, EKV, ENO1, ENO1P, EPB41, EPHX1, F13B, F5,FCGR2A, FCGR2B, FCGR3A, FCHL, FH, FMO3, FMO4, FUCA1, FY, GALE, GBA,GFND, GJA8, GJB3, GLC3B, HF1, HMGCL, HPC1, HRD, HRPT2, HSD3B2, HSPG2,KCNQ4, KCS, KIF1B, LAMB3, LAMC2, LGMD1B, LMNA, LOR, MCKD1, MCL1, MPZ,MTHFR, MTR, MUTYH, MYOC, NB, NCF2, NEM1, NPHS2, NPPA, NRAS, NTRK1,OPTA2, PBX1, PCHC, PGD, PHA2A, PHGDH, PKLR, PKP1, PLA2G2A, PLOD, PPDX,PPTO, PRCC, PRG4, PSEN2, PTOS1, REN, RFX5, RHD, RMD1, RPE65, SCCD,SERPINC1, SJS1, SLC19A2, SLC2A1, SPG23, SPTA1, TALL TNFSF6, TNNT2, TPM3,TSHB, UMPK, UOX, UROD, USH2A, VMGLOM, VWS, WS2B, ABCB11, ABCG5, ABCG8,ACADL, ACP1, AGXT, AHHR, ALMS1, ALPP, ALS2, APOB, BDE, BDMR, BJS, BMPR2,CHRNA1, CMCWTD, CNGA3, COL3A1, COLAA3, COL4A4, COL6A3, CPS1, CRYGA,CRYGEP1, CYP1B1, CYP27A1, DBI, DES, DYSF, EDAR, EFEMP1, EIF2AK3, ERCC3,FSHR, GINGF, GLC1B, GPD2, GYPC, HADHA, HADHB, HOXD13, HPE2, IGKC, IHH,IRS1, ITGA6, KHK, KYNU, LCT, LHCGR, LSFC, MSH2, MSH6, NEB, NMTC, NPHP1,PAFAH1P1, PAX3, PAX8, PMS1, PNKD, PPH1, PROC, REG1A, SAG, SFTPB,SLC11A1, SLC3A1, SOS1, SPG4, SRD5A2, TCL4, TGFA, TMD, TPO, UGT1A@, UV24,WSS, XDH, ZAP70, ZFHX1B, ACAA1, AGS1, AGTR1, AHSG, AMT, ARMET, BBS3,BCHE, BCPM, BTD, CASR, CCR2, CCR5, CDL1, CMT2B, COL7A1, CP, CPO, CRV,CTNNB1, DEM, ETM1, FANCD2, FIR, FOXL2, GBE1, GLB1, GLCLC, GNAI2, GNAT1,GP9, GPX1, HGD, HRG, ITIH1, KNG, LPP, LRS1, MCCC1, MDS1, MHS4, MITF,MLH1, MYL3, MYMY, OPA1, P2RY12, PBXP1, PCCB, POU1F1, PPARG, PROS1,PTHR1, RCA1, RHO, SCAT, SCLC1, SCN5A, SI, SLC25A20, SLC2A2, TF, TGFBR2,THPO, THRB, TKT, TM4SF1, TRH, UMPS, UQCRC1, USH3A, VHL, WS2A, XPC,ZNF35, ADH1B, ADH1C, AFP, AGA, AIH2, ALB, ASMD, BFHD, CNGA1, CRBM, DCK,DSPP, DTDP2, ELONG, ENAM, ETFDH, EVC, Flt, FABP2, FGA, FGB, FGFR3, FGG,FSHMD1A, GC, GNPTA, GNRHR, GYPA, HCA, HCL2, HD, HTN3, HVBS6, IDUA, IF,JPD, KIT, KLKB1, LQT4, MANBA, MLLT2, MSX1, MTP, NR3C2, PBT, PDE6B, PEE1,PITX2, PKD2, QDPR, SGCB, SLC25A4, SNCA, SOD3, STATH, TAPVR1, TYS, WBS2,WFS1, WHCR, ADAMTS2, ADRB2, AMCN, AP3B1, APC, ARSB, B4GALT7, BHR1, C6,C7, CCAL2, CKN1, CMDJ, CRHBP, CSF1R, DHFR, DIAPH1, DTR, EOS, EPD, ERVR,F12, FBN2, GDNF, GHR, GLRA1, GM2A, HEXB, HSD17B4, ITGA2, KFS, LGMDLA,LOX, LTC4S, MAN2A1, MCC, MCCC2, MSH3, MSX2, NR3C1, PCSK1, PDE6A, PFBI,RASA1, SCZD1, SDHA, SGCD, SLC22A5, SLC26A2, SLC6A3, SM1, SMA@, SMN1,SMN2, SPINK5, TCOF1, TELAB1, TGFBI, ALDH5A1, ARG1, AS, ASSP2, BCKDHB,BF, C2, C4A, CDKN1A, COL10A1, COL11A2, CYP21A2, DYX2, EJM1, ELOVL4,EPM2A, ESR1, EYA4, F13A1, FANCE, GCLC, GJA1, GLYS1, GMPR, GSE, HCR, HFE,HLA-A, HLA-DPB1, HLA-DRA, HPFH, ICS1, IDDM1, IFNGR1, IGAD1, IGF2R, ISCW,LAMA2, LAP, LCA5, LPA, MCDR1, MOCS1, MUT, MYB, NEU1, NKS1, NYS2, OA3,ODDD, OFC0, PARK2, PBCA, PBCRA1, PDB1, PEX3, PEX6, PEX7, PKHD1, PLA2G7,PLG, POLH, PPAC, PSORS1, PUJO, RCD1, RDS, RHAG, RP14, RUNX2, RWS, SCA1,SCZD3, SIASD, SOD2, ST8, TAP1, TAP2, TFAP2B, TNDM, TNF, TPBG, TPMT,TULP1, WISP3, AASS, ABCB1, ABCB4, ACHE, AQP1, ASL, ASNS, AUTS1, BPGM,BRAF, C7orf2, CACNA2D1, CCM1, CD36, CFTR, CHORDOMA, CLCN1, CMH6, CMT2D,COL1A2, CRS, CYMD, DFNA5, DLD, DYT11, EEC1, ELN, ETV1, FKBP6, GCK,GHRHR, GHS, GLI3, GPDS1, GUSB, HLXB9, HOXA13, HPFH2, HRX, IAB, IMMP2L,KCNH2, LAMBI, LEP, MET, NCF1, NM, OGDH, OPN1SW, PEX1, PGAM2, PMS2, PON1,PPP1R3A, PRSS1, PTC, PTPN12, RP10, RP9, SERPINE1, SGCE, SHFM1, SHH,SLC26A3, SLC26A4, SLOS, SMAD1, TBXAS1, TWIST, ZWS1, ACHM3, ADRB3, ANK1,CA1, CA2, CCAL1, CLN8, CMT4A, CNGB3, COH1, CPP, CRH, CYP11B1, CYP11B2,DECR1, DPYS, DURS1, EBS1, ECA1, EGI, EXT1, EYA1, FGFR1, GNRH1, GSR,GULOP, HR, KCNQ3, KFM, KWE, LGCR, LPL, MCPH1, MOS, MYC, NAT1, NAT2,NBS1, PLAT, PLEC1, PRKDC, PXMP3, RP1, SCZD6, SFTPC, SGM1, SPG5A, STAR,TG, TRPS1, TTPA, VMD1, WRN, ABCA1, ABL1, ABO, ADAMTS13, AK1, ALAD,ALDH1A1, ALDOB, AMBP, AMCD1, ASS, BDMF, BSCL, C5, CDKN2A, CHAC, CLA1,CMD1B, COL5A1, CRAT, DBH, DNAI1, DYS, DYT1, ENG, FANCC, FBP1, FCMD,FRDA, GALT, GLDC, GNE, GSM1, GSN, HSD17B3, HSN1, IBM2, INVS, JBTS1,LALL, LCCS1, LCCS, LGMD2H, LMX1B, MLLT3, MROS, MSSE, NOTCH1, ORM1,PAPPA, PIP5K1B, PTCH, PTGS1, RLN1, RLN2, RMRP, ROR2, RPD1, SARDH,SPTLC1, STOM, TDFA, TEK, TMC1, TRIM32, TSC1, TYRP1, XPA, CACNB2,COL17A1, CUBN, CXCL12, CYP17, CYP2C19, CYP2C9, EGR2, EMX2, ERCC6, FGFR2,HK1, HPS1, IL2RA, LGI1, LIPA, MAT1A, MBL2, MKI67, MXI1, NODAL, OAT,OATL3, PAX2, PCBD, PEO1, PHYH, PNLIP, PSAP, PTEN, RBP4, RDPA, RET,SFTPA1, SFTPD, SHFM3, SIAL, THC2, TLX1, TNFRSF6, UFS, UROS, AA, ABCC8,ACAT1, ALX4, AMPD3, ANC, APOAL, APOA4, APOC3, ATM, BSCL2, BWS, CALCA,CAT, CCND1, CD3E, CD3G, CD59, CDKNLC, CLN2, CNTF, CPT1A, CTSC, DDB1,DDB2, DHCR7, DLAT, DRD4, ECB2, ED4, EVR1, EXT2, F2, FSHB, FTH1, G6PT1,G6PT2, GIF, HBB, HBBP1, HBD, HBE1, HBG1, HBG2, HMBS, HND, HOMG2, HRAS,HVBS1, IDDM2, IGER, INS, JBS, KCNJ11, KCNJ1, KCNQ1, LDHA, LRP5, MEN1,MLL, MYBPC3, MYO7A, NNO1, OPPG, OPTB1, PAX6, PC, PDX1, PGL2, PGR, PORC,PTH, PTS, PVRL1, PYGM, RAG1, RAG2, ROM1, RRAS2, SAA1, SCA5, SCZD2, SDHD,SERPING1, SMPD1, TCIRG1, TCL2, TECTA, TH, TREH, TSG101, TYR, USH1C,VMD2, VRNI, WT1, WT2, ZNF145, A2M, AAAS, ACADS, ACLS, ACVRL1, ALDH2,AMHR2, AOM, AQP2, ATD, ATP2A2, BDC, CIR, CD4, CDK4, CNA1, COL2A1,CYP27B1, DRPLA, ENUR2, FEOM1, FGF23, FPF, GNB3, GNS, HAL, HBP1, HMGA2,HMN2, HPD, IGF1, KCNA1, KERA, KRAS2, KRT1, KRT2A, KRT3, KRT4, KRT5,KRT6A, KRT6B, KRTHB6, LDHB, LYZ, MGCT, MPE, MVK, MYL2, OAP, PAH, PPKB,PRB3, PTPN11, PXR1, RLS, RSN, SAS, SAX1, SCA2, SCNN1A, SMAL, SPPM,SPSMA, TBX3, TBX5, TCF1, TPI1, TSC3, ULR, VDR, VWF, ATP7B, BRCA2, BRCD1,CLN5, CPB2, ED2, EDNRB, ENUR1, ERCC5, F10, F7, GJB2, GJB6, IPF1, MBS1,MCOR, NYS4, PCCA, RB1, RHOK, SCZD7, SGCG, SLC10A2, SLC25A15, STARP1,ZNF198, ACHM1, ARVD1, BCH, CTAA1, DAD1, DFNB5, EML1, GALC, GCH1, IBGC1,IGH@, IGHC group, IGHG1, IGHM, IGHR, IV, LTBP2, MJD, MNG1, MPD1, MPS3C,MYH6, MYH7, NP, NPC2, PABPN1, PSEN1, PYGL, RPGRIP1, SERPINA1, SERPINA3,SERPINA6, SLC7A7, SPG3A, SPTB, TCL1A, TGM1, TITF1, TMIP, TRA@, TSHR,USHLA, VP, ACCPN, AHO2, ANCR, B2M, BBS4, BLM, CAPN3, CDAN1, CDAN3, CLN6,CMH3, CYP19, CYP1A1, CYP1A2, DYX1, EPB42, ETFA, EYCL3, FAH, FBN1, FES,HCVS, HEXA, IVD, LCS1, LIPC, MY05A, OCA2, OTSC1, PWCR, RLBP1, SLC12A1,SPG6, TPM1, UBE3A, WMS, ABCC6, ALDOA, APRT, ATP2A1, BBS2, CARD15, CATM,CDH1, CETP, CHST6, CLN3, CREBBP, CTH, CTM, CYBA, CYLD, DHS, DNASE1,DPEP1, ERCC4, FANCA, GALNS, GAN, HAGH, HBA1, HBA2, HBHR, HBQ1, HBZ,HBZP, HP, HSD11B2, IL4R, LIPB, MC1R, MEFV, MHC2TA, MLYCD, MMVP1, PHKB,PHKG2, PKD1, PKDTS, PMM2, PXE, SALL1, SCA4, SCNN1B, SCNN1G, SLC12A3,TAT, TSC2, VD1, WT3, ABR, ACACA, ACADVL, ACE, ALDH3A2, APOH, ASPA,AXIN2, BCL5, BHD, BLMH, BRCA1, CACD, CCA1, CCZS, CHRNB1, CHRNE, CMT1A,COL1A1, CORDS, CTNS, EPX, ERBB2, G6PC, GAA, GALK1, GCGR, GFAP, GH1, GH2,GP1BA, GPSC, GUCY2D, ITGA2B, ITGB3, ITGB4, KRT10, KRT12, KRT13, KRT14,KRT14L1, KRT14L2, KRT14L3, KRT16, KRT16L1, KRT16L2, KRT17, KRT9, MAPT,MDB, MDCR, MGI, MHS2, MKS1, MPO, MYO15A, NAGLU, NAPB, NF1, NME1, P4HB,PAFAH1B1, PECAM1, PEX12, PHB, PMP22, PRKAR1A, PRKCA, PRKWNK4, PRP8,PRPF8, PTLAH, RARA, RCV1, RMSA1, RP17, RSS, SCN4A, SERPINF2, SGCA, SGSH,SHBG, SLC2A4, SLC4A1, SLC6A4, SMCR, SOST, SOX9, SSTR2, SYM1, SYNS1,TCF2, THRA, TIMP2, TOC, TOP2A, TP53, TRIM37, VBCH, ATP8B1, BCL2, CNSN,CORD1, CYB5, DCC, F5F8D, FECH, FEO, LAMA3, LCFS2, MADH4, MAFD1, MC2R,MCL, MYP2, NPC1, SPPK, TGFBRE, TGIF, TTR, AD2, AMH, APOC2, APOE, ATHS,BAX, BCKDHA, BCL3, BFIC, C3, CACNA1A, CCO, CEACAM5, COMP, CRX, DBA, DDU,DFNA4, DLL3, DM1, DMWD, E11S, ELA2, EPOR, ERCC2, ETFB, EXT3, EYCL1, FTL,FUT1, FUT2, FUT6, GAMT, GCDH, GPI, GUSM, HB1, HCL1, HHC2, HHC3, ICAM3,INSR, JAK3, KLK3, LDLR, LHB, LIG1, LOH19CR1, LYL1, MAN2B1, MCOLN1, MDRV,MLLT1, NOTCH3, NPHS1, OFC3, OPA3, PEPD, PRPF31, PRTN3, PRX, PSG1, PVR,RYR1, SLC5A5, SLC7A9, STK11, TBXA2R, TGFB1, TNNI3, TYROBP, ADA, AHCY,AVP, CDAN2, CDPD1, CHED1, CHED2, CHRNA4, CST3, EDN3, EEGV1, FTLL1, GDF5,GNAS, GSS, HNF4A, JAG1, KCNQ2, MKKS, NBIA1, PCK1, PI3, PPCD, PPGB, PRNP,THBD, TOP1, AIRE, APP, CBS, COL6A1, COL6A2, CSTB, DCR, DSCR1, FPDMM,HLCS, HPE1, ITGB2, KCNE1, KNO, PRSS7, RUNX1, SOD1, TAM, ADSL, ARSA, BCR,CECR, CHEK2, COMT, CRYBB2, CSF2RB, CTHM, CYP2D6, CYP2D7P1, DGCR, DIA1,EWSR1, GGT1, MGCR, MN1, NAGA, NE2, OGS2, PDGFB, PPARA, PRODH, SCO2,SCZD4, SERPIND1, SLC5A1, SOX10, TCN2, TIMP3, TST, VCF, ABCD1, ACTL1,ADFN, AGMX2, AHDS, AIC, AIED, AIH3, ALAS2, AMCD, AMELX, ANOP1, AR,ARAF1, ARSC2, ARSE, ARTS, ARX, ASAT, ASSP5, ATP7A, ATRX, AVPR2, BFLS,BGN, BTK, BZX, C1HR, CACNA1F, CALB3, CBBM, CCT, CDR1, CFNS, CGF1, CHM,CHR39c, CIDX, CLA2, CLCN5, CLS, CMTX2, CMTX3, CND, COD1, COD2, COL4A5,COL4A6, CPX, CVD1, CYBB, DCX, DFN2, DFN4, DFN6, DHOF, DIAPH2, DKC1, DMD,DSS, DYT3, EBM, EBP, ED1, ELK1, EMD, EVR2, F8, F9, FCP1, FDPSL5, FGD1,FGS1, FMR1, FMR2, G6PD, GABRA3, GATA1, GDI1, GDXY, GJB1, GK, GLA, GPC3,GRPR, GTD, GUST, HMS1, HPRT1, HPT, HTC2, HTR2c, HYR, IDS, IHG1, IL2RG,INDX, IP1, IP2, JMS, KAL1, KFSD, L1CAM, LAMP2, MAA, MAFD2, MAOA, MAOB,MCF2, MCS, MEAX, MECP2, MF4, MGC1, MIC5, MID1, MLLT7, MLS, MRSD, MRX14,MRX1, MRX20, MRX2, MRX3, MRX40, MRXA, MSD, MTM1, MYCL2, MYP1, NDP, NHS,NPHL1, NROB1, NSX, NYS1, NYX, OA1, OASD, OCRL, ODT1, OFD1, OPA2, OPD1,OPEM, OPN1LW, OPN1MW, OTC, P3, PDHA1, PDR, PFC, PFKFB1, PGK1, PGK1P1,PGS, PHEX, PHKA1, PHKA2, PHP, PIGA, PLP1, POF1, POLA, POU3F4, PPMX, PRD,PRPS1, PRPS2, PRS, RCCP2, RENBP, RENS1, RP2, RP6, RPGR, RPS4X, RPS6KA3,RS1, S11, SDYS, SEDL, SERPINA7, SH2D1A, SHFM2, SLC25A5, SMAX2, SRPX,SRS, STS, SYN1, SYP, TAF1, TAZ, TBX22, TDD, TFE3, THAS, THC, TIMM8A,TIM1, TKCR, TNFSF5, UBE1, UBE2A, WAS, WSN, WTS, WWS, XIC, XIST, XK, XM,XS, ZFX, ZIC3, ZNF261, ZNF41, ZNF6, AMELY, ASSP6, AZF1, AZF2, DAZ, GCY,RPS4Y, SMCY, ZFY, ABAT, AEZ, AFA, AFD1, ASAH1, ASD1, ASMT, CCAT, CECR9,CEPA, CLA3, CLN4, CSF2RA, CTS1, DF, DIH1, DWS, DYT2, DYT4, EBR3, ECT,EEF1A1L14, EYCL2, FANCB, GCSH, GCSL, GIP, GTS, HHG, HMI, HOAC, HOKPP2,HRPT1, HSD3B3, HTC1, HV1S, ICHQ, ICR1, ICR5, IL3RA, KAL2, KMS, KRT18,KSS, LCAT, LHON, LIMM, MANBB, MCPH2, MEB, MELAS, MIC2, MPFD, MS, MSS,MTATP6, MTCO1, MTCO3, MTCYB, MTND1, MTND2, MTND4, MTND5, MTND6, MTRNR1,MTRNR2, MTTE, MTTG, MTTI, MTTK, MTTL1, MTTL2, MTTN, MTTP, MTTS1, NAMSD,OCD1, OPD2, PCK2, PCLD, PCOS1, PFKM, PKD3, PRCA1, PRO1, PROP1, RBS,RFXAP, RP, SHOX, SLC25A6, SPG5B, STO, SUOX, THM, and TTD.
 17. Thecomposition of claim 1, wherein said in vitro-synthesized RNA issynthesized by in vitro transcription in a reaction mixture comprising a5′-triphosphate derivative of said pseudouridine-modified nucleoside.18. The composition of claim 1, wherein said in vitro-synthesizedmodified RNA further comprises the modified nucleoside 5-methylcytidine(m⁵C).
 19. The composition of claim 18, wherein said invitro-synthesized modified RNA exhibits a detectable decrease inimmunogenicity to said mammalian cell compared to the same quantity ofin vitro-synthesized unmodified RNA that exhibits the same sequenceexcept with uridine in place of said modified nucleoside selected fromthe group consisting of (i) 1-methypseudouridine (m¹Ψ) and (ii)pseudouridine (Ψ), and cytidine in place of said 5-methylcytidinemodified nucleoside.
 20. The composition of claim 18, wherein said invitro-synthesized RNA is synthesized by in vitro transcription in areaction mixture comprising a 5′-triphosphate derivatives of bothpseudouridine and 5-methylcytidine modified nucleosides.
 21. Thecomposition of claim 18, wherein said composition is present in amammalian cell that is in culture, in a tissue, or in vivo in a mammal.